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WO2025166202A1 - Lipid nanoparticle compositions comprising sialic acid derivatives and the uses thereof - Google Patents

Lipid nanoparticle compositions comprising sialic acid derivatives and the uses thereof

Info

Publication number
WO2025166202A1
WO2025166202A1 PCT/US2025/014085 US2025014085W WO2025166202A1 WO 2025166202 A1 WO2025166202 A1 WO 2025166202A1 US 2025014085 W US2025014085 W US 2025014085W WO 2025166202 A1 WO2025166202 A1 WO 2025166202A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
mol
previous
nanoparticle
mir
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/014085
Other languages
French (fr)
Inventor
Alec FREYN
Mohindra Seepersaud
Frank PICKARD
Ellalahewage S. Kumarasinghe
Junyong KIM
Yue HUI
Karthik Ramachandran
Jonathan Hoggatt
Youmna KFOURY
David EASTERHOFF
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ModernaTx Inc
Original Assignee
ModernaTx Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ModernaTx Inc filed Critical ModernaTx Inc
Publication of WO2025166202A1 publication Critical patent/WO2025166202A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A61K31/7024—Esters of saccharides
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H15/14—Acyclic radicals, not substituted by cyclic structures attached to a sulfur, selenium or tellurium atom of a saccharide radical
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
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    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • Lapid nanoparticles have proven effective as transport vehicles into cells and/or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids. Though a variety of such lipid-containing nanoparticles have been demonstrated, improvements in safety, efficacy, and specificity are still needed.
  • the present disclosure provides a sialic acid lipid of Formula (SA-I): or a salt or ionized form thereof.
  • the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid described herein.
  • the present disclosure provides a population of lipid nanoparticles comprising a sialic acid lipid, an ionizable lipid, and a structural lipid, wherein the sialic acid lipid of Formula (SA-1):
  • the present disclosure provides methods of preparing the lipid nanoparticle and the population of lipid nanoparticles described herein.
  • the present disclosure provides pharmaceutical compositions comprising the lipid nanoparticle, pharmaceutical compositions comprising the population of lipid nanoparticles described herein, and methods of preparing the pharmaceutical compositions.
  • the disclosure provides methods of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
  • HSPC hematopoietic stem and progenitor cell
  • the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in delivering therapeutic agents to hematopoietic stem and progenitor cells (HSPC) in subjects.
  • HSPC hematopoietic stem and progenitor cells
  • the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of medicaments for delivering therapeutic agents to hematopoietic stem and progenitor cells (HSPC) in subjects.
  • the disclosure provides methods of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
  • EPC erythroid progenitor cell
  • the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in delivering therapeutic agents to erythroid progenitor cells (EPC) in subjects.
  • EPC erythroid progenitor cells
  • the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of medicaments for delivering therapeutic agents to erythroid progenitor cell (EPC) in subjects.
  • EPC erythroid progenitor cell
  • the disclosure provides methods of delivering a therapeutic agent to a myeloid cell in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
  • the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition for use in delivering therapeutic agents to myeloid cells in subjects,
  • the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition in the manufacture of medicaments for delivering therapeutic agents to myeloid cells in subjects.
  • the disclosure provides methods of delivering a therapeutic agent to a lymophoid cell in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
  • the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition for use in delivering therapeutic agents to lymophoid cells in subjects.
  • the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition in the manufacture of medicaments for delivering therapeutic agents to lymophoid cells in subjects.
  • the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in treating or preventing diseases or disorders in subjects.
  • the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of a medicaments for treating or preventing diseases and disorders in subjects.
  • the present disclosure provides nanoparticles prepared by the processes of preparing nanoparticles provided herein.
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein the mRNA comprises an open reading frame encoding a cancer antigen or an infectious disease antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (li) the vaccine comprises a mRNA comprising an open reading frame encoding a cancer antigen or an infectious disease antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises (i-i) a sialic acid disclosed herein (e.g., Compound No. 9), (i-ii) an ionizable lipid disclosed herein, (i-iii) a structural lipid disclosed herein, (i-iv) a phospholipid disclosed herein, and (i-v) a PEG-lipid disclosed herein, and (ii) the mRNA comprises an open reading frame encoding a cancer antigen or an infectious disease antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a method of reducing susceptibility to or symptoms of an infectious disease in a subject, comprising administering a vaccine disclosed herein to the subject, wherein the vaccine comprises an mRNA comprising an open reading frame encoding an infectious disease antigen.
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in a method of reducing susceptibility to or symptoms of an infectious disease in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRN A comprises an open reading frame encoding an infectious disease antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in the preparation of a medicament for reducing susceptibility to or symptoms of an infectious disease in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding an infectious disease antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides the use of a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding an infectious disease antigen, for reducing susceptibility to or symptoms of an infectious disease in a subject.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a method treating cancer in a subject, comprising administering a vaccine disclosed herein to the subject, wherein the mRNA comprises an open reading frame encoding a cancer antigen.
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in a method of treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in the preparation of a medicament for treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
  • mRNA messenger ribonucleic acid
  • the present, disclosure provides the use of a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
  • mRNA messenger ribonucleic acid
  • the present disclosure provides a composition comprising (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle disclosed herein.
  • a nucleic acid comprising a sequence encoding a CRISPR nuclease
  • gRNA guide RNA
  • the present disclosure provides a composition
  • a composition comprising: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle comprising (iii-i) an ionizable lipid disclosed herein, (iii-ii) a PEG lipid disclosed herein, (iii-iii) a structural lipid disclosed herein, (iii-iv) a phospholipid disclosed herein, and (iii- v) a sialic acid lipid disclosed herein (e.g., Compound No. 1).
  • the present disclosure provides a method of editing a sequence of a target nucleic acid in a cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle disclosed herein.
  • gRNA guide RNA
  • the present disclosure provides a method of editing a sequence of a target nucleic acid in a cell, preferably a human cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle comprising (iii-i) an ionizable lipid disclosed herein, (iii-ii) a PEG lipid disclosed herein, (iii-ii) a structural lipid disclosed herein, (iii-iv) a phospholipid disclosed herein, and (iii-v) a sialic acid lipid disclosed herein (e.g., Compound No. 1).
  • the present disclosure provides a method of delivering a polypeptide to a cell, preferably a human cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding the polypeptide; and (ii) a lipid nanoparticle comprising (ii-i) an ionizable lipid disclosed herein, (ii-ii) a PEG lipid disclosed herein, (ii-iii) a structural lipid disclosed herein, (ii-iv) a phospholipid disclosed herein, and (iii-v) a sialic acid lipid disclosed herein.
  • the polypeptide comprises a therapeutic agent.
  • FIG. 1 shows a flow-chart of PIPA process where sialic acid lipids were incorporated in the lipid stock solution.
  • FIG. 2 shows a flow-chart of PIPA process where sialic acid lipids were added in the PI stage.
  • FIG. 3 shows a HPLC chromatogram showing a distinct peak corresponding to compound 1 in an LNP produced by the process shown in FIG. 2.
  • FIG. 4 shows Octet Biolayer Interferometry (BLI) characterization of sialic acid LNPs.
  • Human CD-33 is immobilized on NT A probes. All sialic acid containing LNPs showed significant binding to CD-33 compared to the control (bottom-most line).
  • FIG. 5 is a plot showing the percent of CD33 insertions/deletions (indels) in mouse bone marrow hematopoietic stem and progenitor cells after intravenous administration of sialic acid LNP containing spCas9 mRNA and an sgRNA targeting mouse CD33 to C57/B16 mice, with or without hematopoietic stem cell mobilization (M).
  • Cells were lineage negative (lin-), and ckit receptor tyrosine kinase positive (ckit+).
  • FIGS. 6A-6C show the percentage of cells expressing green lantern after intravenous delivery of green lantern mRNA using the indicated sialic acid LNP to humanized female mice (Hu-CD34+NSG-SGM3).
  • FIG. 6A shows expression of green lantern in my eloid progenitors cells (hCD45+, Lineage negative, CD34+ and CD38+).
  • FIG. 6B shows green lantern expression in hematopoietic stem and progenitor cells (HSPCs) ( HCD45+, Lineage negative, CD34+ and CD38- ).
  • HSPCs hematopoietic stem and progenitor cells
  • FIG. 7 is a diagram showing sialic acid LNP administration and sample collection for the experiments described in Example 8.
  • FIG. 8 is a plot showing average cell viability for cells collected from groups of mice administered sialic acid LNP as described in Example 8.
  • FIG. 9 is a plot showing average cell counts for cells collected from the spleens of the groups of mice administered sialic acid LNP as described in Example 8.
  • FIG. 10 is a plot showing the percentage of CD4+ T cells expressing, from left to right for each set of bars, Thl cytokines (IFNy, TNFa and IL-2), and Th2 cytokines (IL-4, IL-5 and IL- 15) for mice administered sialic acid containing LNPs as described in Example 8.
  • Thl cytokines IFNy, TNFa and IL-2
  • Th2 cytokines IL-4, IL-5 and IL- 15
  • FIG. 11 is a plot showing the percentage of CD8+ T cells expressing, from left to right for each set of bars, CD 107 ⁇ , IFN ⁇ , TNF ⁇ and IL-2 for mice administered sialic acid containing LNPs as described in Example 8.
  • FIG. 12 is a plot showing influenza-specific antibody titers against influenza virus A/Sydney/5/2021 using sera from mice administered sialic acid containing LNPs as described in Example 8.
  • FIG. 13 is a plot showing total H1 Sydney IgG Titers at days 21 and 36 as described in Example 8.
  • FIG. 14 is a series of plots showing post-boost cytokine responses (Luminex, 6 hours post boost) for mice administered sialic acid containing LNPs as described in Example 8. Geometric mean responses and geometric 95% CI are shown for each group.
  • FIG. 15 is a series of plots showing post-boost, cytokine responses (Luminex, 6 hours post boost) for mice administered sialic acid containing LNPs as described in Example 8. Geometric mean responses and geometric 95% CI are shown for each group.
  • the present disclosure provides a sialic acid lipid of Formula (SA-I): or a salt or ionized form thereof, wherein:
  • R is C 13-20 alkyl or C 13-20 alkenyl
  • R’ is C 13-20 alkyl or C 13-20 alkenyl
  • L is - (C 3-8 alkylene)-T-* or -(C 3-8 heteroalkylene)-T-*, wherein the C 3-8 alkylene or C 3-8 heteroalkylene is optionally substituted with one or more oxo;
  • T is -CH 2 -, -O-, -S-, or -NH-;
  • R 1 is -OR 1 or -NR 1 -C( ())-R r ”;
  • R’ is H or C 1-6 alkyl
  • R '' is H or C 1-6 alkyl
  • R 1''' is C 1-6 alkyl
  • the sialic acid lipid is of Formula (SA-II):
  • the sialic acid lipid is of Formula (SA- IF):
  • the sialic acid lipid is of Formula (SA-II”):
  • the sialic acid lipid is of Formula (SA-III):
  • the sialic acid lipid is of Formula (SA-IV):
  • the sialic acid lipid is of Formula (SA-V): (SA-V) or a salt or ionized form thereof.
  • the sialic acid lipid is of Formula (SA-V-i):
  • the sialic acid lipid is of Formula (SA-V-ii):
  • the sialic acid lipid is of Formula (SA-VI):
  • the sialic acid lipid is of Formula (SA-VI-i):
  • the sialic acid lipid is of Formula (SA-VI-ii):
  • the present disclosure provides a lipid comprising a diacylated propylene glycol moiety, a phosphate moiety, a PEG moiety, and a sialic acid moiety.
  • R is C 13-20 alkyl. [0073] In some embodiments, R is C 15-20 alkyl. [0074] In some embodiments, R is C 13-15 alkyl. [0075] In some embodiments, R is C 16-18 alkyl. [0076] In come embodiments, R is C 13 alkyl.
  • R is C 14 alkyl.
  • R is C 15 alkyl.
  • R is C 16 alkyl.
  • R is C 17 alkyl.
  • R is C 18 alkyl.
  • R is C 19 alkyl
  • R is C 20 alkyl
  • R’ is C 13-20 alkyl.
  • R’ is C 15-20 alkyl.
  • R’ is C 13-15 alkyl.
  • R’ is C 16-18 alkyl. [0088] In come embodiments, R’ is C 13 alkyl. [0089] In come embodiments, R’ is C 14 alkyl. [0090] In come embodiments, R’ is C 15 alkyl. [0091] In some embodiments, R’ is C 16 alkyl. [0092] In some embodiments, R’ is C 17 alkyl.
  • R’ is C 18 alkyl. [0094] In some embodiments, R’ is C 19 alkyl. [0095] In some embodiments, R 1 is C 20 alkyl. [0096] In some embodiments, R is C 15-20 alkenyl. [0097] In some embodiments, R is C 13-15 alkenyl [0098] In some embodiments, R is C 16-18 alkenyl.
  • R is C 15 alkenyl. [0100] In some embodiments, R is C 16 alkenyl.
  • R is C 17 alkenyl. [0102] In some embodiments, R is C 18 alkenyl.
  • R is C 19 alkenyl.
  • R is C 20 alkenyl.
  • R’ is C 15-20 alkenyl.
  • R’ is C 13-15 alkenyl.
  • R’ is C 16-18 alkenyl.
  • R’ is C 15 alkenyl.
  • R’ is C 16 alkenyl.
  • R’ is C 17 alkenyl.
  • R’ is C 18 alkenyl.
  • R’ is C 19 alkenyl.
  • R’ is C 20 alkenyl.
  • n 40 to 50.
  • n 41-45.
  • n is 42-44.
  • n 43-44.
  • n 40.
  • n 41
  • n is 42.
  • n 43.
  • n 44.
  • n 45
  • n 46.
  • n 47.
  • n 48.
  • n 49.
  • n is 50.
  • X is a metal cation
  • X is an alkali metal cation.
  • X is a sodium cation
  • X is a lithium cation
  • X + is a potassium cation.
  • X + is an ammonium cation.
  • L comprises a C 3-8 alkylene moiety.
  • L is -(C 3-8 alkylene)-X’-*, wherein:
  • R is H or C 1-6 alkyl; and the C 1-10 alkylene is optionally substituted with one or more oxo groups.
  • T is -CH 2 -.
  • T is -O-.
  • T is -S-.
  • T is -NH-.
  • the alkylene is linear.
  • the alkylene is branched.
  • L is --(C 3-8 alkylene)-O)-*.
  • L is -(C 3-8 alkylene)-S-*.
  • L is (C 3-8 alkylene)-NH-*.
  • L comprises a C 3-8 heteroalkylene moiety.
  • L is --(C 3-8 heteroalkylene)-X’-*, wherein:
  • T is -CH 2 -, -O-, -S-, or -NR-, wherein R is H or C 1-6 alkyl; and the C 1 -10 alkylene is optionally substituted with one or more oxo groups.
  • the heteroalkylene is linear.
  • the heteroalkylene is branched.
  • L is heteroalkylene)-O-* .
  • L is heteroalkylene)-S-*.
  • L is heteroalkylene)-NH- * .
  • L is , wherein indicates attachment to -La-Sa.
  • L is
  • L is , wherein indicates attachment to -La-Sa.
  • L is N
  • L is , wherein indicates attachment to -La-Sa.
  • L is wherein indicates attachment to -La-Sa.
  • L is , wherein indicates attachment to -La-Sa.
  • La is a lactosyl moiety is derived from lactose.
  • the lactosyl moiety is a derivative of lactose.
  • the present disclosure contemplates the use of lactose, lactosamme, or N-acetyl lactosamme,
  • lactosyl moieties wherein an atom from the L moiety, e.g, a heteroatom represented by X’, substitutes for a hydroxyl group in lactose.
  • the present disclosure contemplates the use of lactosyl moieties wherein the hydroxyl group at the anomeric position of lactose is replaced by a heteroatom from the L moiety.
  • the lactosyl moiety comprises lactose.
  • the lactosyl moiety comprises lactosamme.
  • the lactosyl moiety comprises N-acetyl lactosamine.
  • La is , wherein indicates attachment to L indicates attachment to -Sa.
  • La is . wherein indicates attachment to L indicates attachment to -Sa,
  • La is wherein indicates attachment indicates attachment to -Sa. [0168] In some embodiments. La is , wherein indicates attachment to
  • La is N-[0169] In some embodiments.
  • La is , wherein indicates attachment to L and indicates attachment to -Sa.
  • La is , wherein indicates attachment to L and indicates attachment to -Sa. [0172] In some embodiments, La is , wherein indicates attachment to L indicates attachment to -Sa.
  • La is , wherein indicates attachment to
  • La is , wherein indicates attachment to
  • La is , wherein indicates attachment to
  • La is wherein indicates attachment to L and indicates attachment to -Sa.
  • Sa is a sialic acid moiety comprising neuraminic acid.
  • the sialic acid comprises a derivative of neuraminic acid.
  • the sialic acid comprises N-glycolneuramimc acid.
  • the sialic acid comprises 2- keto-3-deoxynonic acid.
  • the sialic acid comprises N-acylated neuraminic acid.
  • Sa is indicates attachment to -
  • Sa is wherein indicates attachment to the lactosyl moiety.
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is [0183] In some embodiments, -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • -La-Sa is
  • the sialic acid lipid is selected from the compounds described in Table SA-1, salts thereof, and ionized forms thereof.
  • the sialic acid lipid is selected from the compounds described in Table SA-1 and salts thereof
  • the sialic acid lipid is selected from the compounds described in Table SA-1, and ionized forms thereof.
  • the sialic acid lipid is selected from the compounds described in Table SA-1.
  • the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid disclosed herein.
  • the lipid nanoparticle further comprises an ionizable lipid.
  • the lipid nanoparticle further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof.
  • the lipid nanoparticle comprises a sialic acid lipid disclosed herein, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the present disclosure provides a population of lipid nanoparticles comprising a sialic acid lipid disclosed herein.
  • the population of lipid nanoparticles further comprises an ionizable lipid.
  • the population of lipid nanoparticles further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof.
  • the population of lipid nanoparticles comprises a sialic acid lipid disclosed herein, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the present disclosure provides lipid nanoparticles and populations of lipid nanoparticles comprising a sialic acid lipid, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
  • the LNPs of the present disclosure may facilitate delivery of nucleic acid payloads to certain cell types or when expression of a protein of interest is desired (e.g., a polypeptide to which an immune response is desired or polypeptide for therapeutic expression) to improve the quality of immune response to an antigen or to dampen the immune response to a therapeutic.
  • a protein of interest e.g., a polypeptide to which an immune response is desired or polypeptide for therapeutic expression
  • the LNPs disclosed herein that, incorporate Compound No. 9 can lead to reduced Th2 cytokine production (IL-4, IL-5 and IL- 15) while maintaining Thl cytokine production (IFNy, TNFa and 11,-2) and CD4+ and CD8+ T cell activation.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to human CD33+ hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to CD169+ marginal Macrophages.
  • LNPs comprising Compound No. 9 are suitable for delivery of nucleic acids polypeptides to promote an immune response (e.g., an antigen as described herein).
  • the LNPs comprising Compound No. 9 improve the quality of the immune response.
  • LNPs comprising Compound. No. 9 can improve the quality of an immune response to a. vaccine.
  • LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a therapeutic polypeptide when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 1 show reduced immune response compared to LNPs that do not comprise Compound No. 1.
  • LNPs comprising Compound No. 9 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 9 show' reduced immune response compared to LNPs that do not comprise Compound No. 9.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to myeloid progenitor cells, e.g. 11CD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a. therapeutic polypeptide to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
  • a. therapeutic polypeptide to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a therapeutic polypeptide to hematopoietic stem and progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
  • LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a. therapeutic polypeptide to erythroid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to erythroid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acid encoding CRISPR protein and/or gRNA to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery' of one or more nucleic acids encoding a CRISPR protein and/or gRNA to hematopoietic stem and. progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
  • the LNPs comprising sialic acid lipids may result in reduced cytokine secretion, reduced inflammatory responses, increased targeting to bone marrow resident HSPCs, differential targeting of myeloid subsets, and enhanced protein production in the liver, as compared to LNPs of different composition.
  • LNPs comprising sialic acid lipids formulated with sialic acid lipid added in the lipid stock solution may generally be small in size.
  • LNPs that the LNPs comprising sialic acid lipids may bind to human CD-33 more strongly than LNPs of different formulation.
  • the LNPs comprising sialic acid lipids may effectuate protein expression in HSPCs (e.g., Lin-cKit+Scal+ cells).
  • LNPs comprising sialic acid lipids may effectuate protein expression in Lin-cKit+Scal- cells (LK cells).
  • LNPs comprising sialic acid lipids may effectuate protein expression in erythroid progenitor cells.
  • LNPs comprising sialic acid lipids may effectuate protein expression in long-term HSCs (LT-HSCs, e.g., Lin- cKit+Scal+CD150+CD48-cells).
  • LT-HSCs long-term HSCs
  • the process by which the LNPs comprising sialic acid lipids are made may play a role in the ability of the LNPs to deliver mRNAs to bone marrow.
  • LNPs comprising sialic acid, lipids may effectuate lower Seal expression (e.g., no significant Seal expression) relative to the Seal expression effectuate by LNPs of different composition.
  • LNPs comprising sialic acid lipids may efficiently target cells such as HSPCs without activating an immune response, e.g., an inflammatory response measured by way of Seal expression, within said cells. It is believed that LNPs of alternative composition that are equivalently efficient at targeting said cells would, in contrast to LNPs comprising sialic acid lipids, activate an immune response, e.g., an inflammatory response measured by way of Seal expression, within said cells.
  • the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid, an ionizable lipid, and a structural lipid, wherein the sialic acid lipid is of Formula (SA-I).
  • a phospholipid useful or potentially useful in the present invention is an anionic phospholipid.
  • the population of lipid nanoparticles comprises between about 0.1 mol % to about 5 mol %, about 0.1 mol % to about 4 mol %, about 0.1 mol % to about 3 mol %, about 0.1 mol % to about 2 mol %, about 0.2 mol % to about 2 mol %, about 0.4 mol % to about 1.5 mol % of the sialic acid lipid, or about 0.4 mol% to about 1 mol% of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0. 1 mol % to about 5 mol % of the sialic acid lipid.
  • the population of lipid nanoparticles comprises between about 0.1 mol % to about 4 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 3 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 2 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.2 mol % to about 2 mol % of the sialic acid lipid.
  • the population of lipid nanoparticles comprises between about 0.4 mol % to about 1.5 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.4 mol % to about 1 mol % of the sialic acid lipid.
  • the population of lipid nanoparticles comprises about 0.1 mol %, about 0.2 mol %, about 0.3 mol %, about 0.4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, or about 2.0 mol % of the sialic acid lipid.
  • the population of lipid nanoparticles comprises about 0.5 mol % of the sialic acid, lipid. In some embodiments, the population of lipid, nanoparticles comprises about 0.6 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.7 mol % of the sialic acid lipid. In some embodiments, the population of lipid, nanoparticles comprises about 0.8 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.9 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 1.0 mol % of the sialic acid lipid.
  • the ionizable lipid is compound 1-301, compound II-6, 1-25, or 1-18. In some embodiments, the ionizable lipid is compound 1-301 or compound II-6,
  • the ionizable lipid is compound 1-301.
  • Compound 1-301 is a compound of the formula. or a salt thereof.
  • the ionizable lipid is compound II-6.
  • Compound II-6 is a compound of the formula or a salt thereof.
  • the ionizable lipid is 1-18.
  • 1-18 is a compound of the formula or a salt thereof.
  • the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of the ionizable lipid. In some embodiments, the population of lipid nanoparticles comprises about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, or about 50 mol % of the ionizable lipid.
  • the population of lipid nanoparticles comprises about 30 mol % to about 50 mol % of the ionizable lipid (e.g., compound 1-301, compound 1-18, or compound II-6). In some embodiments, the population of lipid nanoparticles comprises about 35 mol % to about 45 mol % of the ionizable lipid (e.g., compound 1-301, compound 1-18, or compound II-6).
  • the population of lipid, nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound 301. In some embodiments, the population of lipid nanoparticles comprises about. 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 47 mol %, or about 50 mol % of compound 1-301.
  • the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound 301 .
  • the population of lipid, nanoparticles comprises about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 47 mol %, or about 50 mol % of compound 1-18.
  • the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound II-6. In some embodiments, the population of lipid nanoparticles comprises about 30 mol %, about 35 mol %, about. 40 mol %, about 45 mol %, about 47 mol %, or about. 50 mol % of compound II-6.
  • the structural lipid is cholesterol
  • the population of lipid nanoparticles comprises about 15 mol % to about 50 mol %, about 20 mol % to about 50 mol %, about 25 mol % to about 50 mol %, about. 30 mol % to about 50 mol %, about 35 mol % to about 50 mol %, about 40 mol % to about 50 mol %, or about 45 mol % to about 50 mol % of the structural lipid (e.g., cholesterol).
  • the structural lipid e.g., cholesterol
  • the population of lipid nanoparticles comprises about 15 mol % to about 45 mol %, about 20 mol % to about 45 mol %, about 25 mol % to about 45 mol %, about 30 mol % to about 45 mol %, about 35 mol % to about 45 mol %, or about 40 mol % to about 45 mol % of the structural lipid (e.g., cholesterol).
  • the structural lipid e.g., cholesterol
  • the population of lipid nanoparticles comprises about 15 mol % to about 40 mol %, about 20 mol % to about 40 mol %, about 25 mol % to about 40 mol %, about 30 mol % to about 40 mol %, or about 35 mol % to about 40 mol % of the structural lipid, (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 20 mol % to about 45 mol % of the structural lipid (e.g;, cholesterol).
  • the population of lipid nanoparticles comprises about 20 mol % to about 40 mol % of the structural lipid (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 30 mol % to about 40 mol % of the structural lipid, (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 39 mol%, about 40 mol %, about. 45 mol %, or about 50 mol % of the structural lipid (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 35 mol % of the structural lipid (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 40 mol % of the structural lipid (e.g., cholesterol).
  • the population of lipid nanoparticles comprises about 45 mol % of the structural lipid (e.g., cholesterol).
  • the population of lipid nanoparticles comprises a phospholipid. In some embodiments, it comprises about 10 mol % to about 30 mol %, about 15 mol % to about 25 mol %, or about 15 mol % to about 20 mol % of the phospholipid. In some embodiments, it comprises about 10 mol %, about 15 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 25 mol %, or about 30 mol % of the phospholipid. In some embodiments, the phospholipid is DMPS , DSPC, DOPE, DOPC, POPE, or POPC.
  • the population of lipid nanoparticles comprises a phospholipid that is DSPC, wherein the population of lipid nanoparticles comprises about 10 mol % to about 30 mol %, about. 10 mol % to about 25 mol %, about 10 mol % to about 20 mol %, about. 15 mol % to about 25 mol %, or about 15 mol % to about 20 mol % of the DSPC.
  • the population of lipid nanoparticles comprises about 10 mol % to about 25 mol % of the DSPC.
  • the population of lipid nanoparticles comprises about 10 mol % to about 20 mol % of the DSPC.
  • the population of lipid, nanoparticles comprises about 15 mol % to about 25 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 15 mol % to about 20 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprise about 10 mol % to about 15 mol % of the DSPC.
  • the population of lipid nanoparticles comprises a phospholipid that is DSPC, wherein the population of lipid nanoparticles comprises about 10 mol %, about 11 mol %, about 15 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 25 mol %, or about 30 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 10 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 20 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 22 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 25 mol % of the DSPC.
  • the population of lipid nanoparticles is free of PEG- lipid.
  • the population of lipid nanoparticles further comprises a PEG lipid.
  • the population of lipid nanoparticles comprises about 0.5 mol % to about 10 mol %, about 0.5 mol % to about 5 mol %, about 0.5 mol% to about 3 mol%, about 1 mol % to about 5 mol %, or about 1 mol % to about 3 mol % of the PEG lipid.
  • the PEG lipid is PL-02.
  • PL-02 refers to a polymer of the formula: or a salt thereof, wherein r is 45.
  • the number of repeating units indicated, in the structure of a polymer refers to the average number of repeating units (a.k.a., average degree of polymerization).
  • r is an integer from about 35 to about 55.
  • the population of lipid nanoparticles comprises about 1 mol % to about 5 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about 3 mol % to about 5 mol % of the PEG lipid (e.g., PL-02).
  • the population of lipid nanoparticles comprises about 0.5 mol %, about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, or about 5 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about. 3 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about 4 mol % of the PEG lipid (e.g., PL-02).
  • the population of lipid nanoparticles comprises about 5 mol % of the PEG lipid (e.g., PL-02).
  • the population of lipid nanoparticles has a. pH value lower than the pKa value of the ionizable lipid. In some embodiments, it has a pH value of about 4.0+2.0, about 4.0+1.5, about 4.0+1.4, about 4.0+ 1.3, about 4.0+1.2, about 4.0+1.1, about 4.0+1.0, about 4.0+0.9, about 4.0+0.8, about 4.0+0.7, about 4.0+0.6, about 4.0+0.5, about 4.0+0.4, about 4.0+0,3, about 4.0+0.2, or about 4.0+0.1.
  • it has a pH value of about 5.0+2.0, about 5.0+1.5, about 5.0+1.4, about 5.0+1.3, about 5.0+1.2, about 5.0+1. 1, about 5.0+1.0, about 5.0+0.9, about 5.0+0.8, about 5.0+0.7, about 5.0+0.6, about 5.0+0.5, about 5.0+0.4, about 5.0+0.3, about 5.0+0.2, or about 5.0+0.1.
  • the population of lipid nanoparticles has a pH value higher than the pKa value of the ionizable lipid. In some embodiments, it has a pH value of about 8.0+2.0, about 8.0+1.5, about 8.0+1.4, about 8.0+1.3, about 8.0 ⁇ 1.2, about 8.0+1 .1 , about 8.0+1 .0, about 8.0+0.9, about 8.0+0.8, about 8.0+0.7, about 8.0+0.6, about 8.0+0.5, about 8.0+0.4, about 8.0+0.3, about 8.0+0.2, or about 8.0+0.1.
  • the population of lipid nanoparticles has a pH value of about 9.0+3.0, about 9.0+2.0, about 9.0+1.5, about 9.0+1.4, about 9.0+1.3, about 9.0+1.2, about 9.0+1.1 , about. 9.0+1 .0, about 9.0+0.9, about 9.0+0.8, about 9.0+0.7, about 9.0+0.6, about 9.0+0.5, about 9.0+0.4, about 9.0+0.3, about 9.0+0.2, or about 9.0+0. 1.
  • the population of lipid nanoparticles has a pH value of about 12.0+2.0, about 12.0+1.5, about 12.0+1.4, about 12.0+1.3, about 12.0+1.2, about 12.0+1.1, about 12.0+1.0, about 12.0+0.9, about. 12.0+0.8, about 12.0+0.7, about 12.0+0.6, about 12.0+0.5, about 12.0+0.4, about 12.0+0.3, about 12.0+0.2, or about 12.0+0.1.
  • the population of lipid nanoparticles has a zeta potential between about -40m V to about - 1 mV, about -40m V to about -5m V, about -30mV to about -5m V, about -20m V to about -5m V, about -40m V to about -10mV, about -30mV to about -10mv, or about -20m V to about -10m V when measured in 0.1N PBS at pH 7.5.
  • the population of lipid nanoparticles has a zeta potential between about -20m V to about -10m V when measured in 0.1N PBS at pH 7.5.
  • the population of lipid nanoparticles is free of therapeutic agent. [0255] In some embodiments, the population of lipid nanoparticles further comprises a therapeutic agent. In some embodiments, the therapeutic agent is an mRNA.
  • Embodiments of the present disclosure are directed to pharmaceutical compositions comprising the population of lipid nanoparticles described herein and one or more pharmaceutically acceptable carriers or excipients. Some embodiments are directed, to methods of preparing the pharmaceutical compositions.
  • Embodiments of the present disclosure are directed to a method of preparing the population of lipid nanoparticles as described herein.
  • Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
  • HSPC hematopoietic stem and progenitor cell
  • Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described, herein for use in delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject.
  • HSPC hematopoietic stem and progenitor cell
  • Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject.
  • HSPC hematopoietic stem and progenitor cell
  • Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
  • EPC erythroid progenitor cell
  • Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to erythroid progenitor cells (EPC) in a subject.
  • EPC erythroid progenitor cells
  • Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to an erythroid progenitor cells (EPC) in a subject.
  • EPC erythroid progenitor cells
  • Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a myeloid cell in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
  • Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to myeloid cells in a subject.
  • Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a therapeutic agent to myeloid cells in a subject.
  • Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to myeloid cells in a subject.
  • Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a lymphoid cell in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
  • Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to lymphoid cells in a subject.
  • Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a therapeutic agent to lymphoid cells in a subject.
  • the subject LNPs can be used to deliver nucleic acid molecules encoding polypeptides against which an immune response is not desired.
  • the reduced levels of cytokine production in subjects to which these LNPs are administered make them particularly desirable delivery vehicles, e.g., for therapeutic protein expression.
  • the subject is human.
  • an empty lipid nanoparticle solution may be prepared by Process 1, Process 1 comprising: i) a nanoprecipitation step, comprising: i-a) a mixing step, comprising mixing a lipid solution comprising an ionizable lipid, a structural lipid, and a phospholipid, with a first aqueous buffer solution, thereby forming an intermediate empty-iipid nanoparticle solution (intermediate empty-LNP solution) comprising an intermediate empty lipid nanoparticle (intermediate empty LNP), i-b) a holding step, comprising holding the intermediate empty-LNP solution for a residence time, and i-c) a diluting step, comprising adding a diluting solution comprising a second aqueous buffer solution to the intermediate empty-LNP solution, thereby forming the empty-LNP solution comprising an empty LNP, wherein the lipid solution, the aqueous buffer solution, and/or the diluting solution comprises a. phosphatid
  • a loaded lipid nanoparticle solution may be prepared by Process 1, Process 1 further comprising:
  • a nanoprecipitation step comprising: i-a) a mixing step, comprising mixing a lipid solution comprising an ionizable lipid, a structural lipid, and a phospholipid, with a first aqueous buffer solution, thereby forming an intermediate empty-iipid nanoparticle solution (intermediate empty-LNP solution) comprising an intermediate empty lipid nanoparticle (intermediate empty LNP): i-b) a holding step, comprising holding the intermediate empty-LNP solution for a residence time; and i-c) a diluting step, comprising adding a diluting solution comprising a second aqueous buffer solution to the intermediate empty-LNP solution, thereby forming the empty-LNP solution comprising an empty LNP, iii) mixing a nucleic acid solution comprising a nucleic acid with the empty-LNP solution, thereby forming the loaded-LNP solution comprising a loaded lipid nanoparticle (loaded LNP), wherein the lipid solution
  • the lipid solution comprises the phosphatidylserine phospholipid.
  • the aqueous buffer solution comprises the phosphatidylserine phospholipid.
  • the diluting solution comprises the phosphatidylserine phospholipid.
  • the empty LNP comprises the phosphatidylserine phospholipid.
  • the mixing step is performed with a first aqueous buffer solution having a pH higher than the pKa of the ionizable lipid.
  • the mixing step is performed at a pH of 4.0 to 12.0.
  • the mixing step is performed at a pH of 12.0 ⁇ 2.0, 12.0 : 1 .5, 12.0 ⁇ 1.0, 12.0 ⁇ 0.9, 12.0 ⁇ -0.8, 12.0 ⁇ 0.7, 12.0 ⁇ 0.6, I2.0 ⁇ 0.5, 12.0 ⁇ 0.4, 12.0 ⁇ 0.3, 12.0 ⁇ 0.2, or 12.0 ⁇ 0.1.
  • the lipid solution comprises one or more phosphatidylserine phospholipid.
  • the phosphatidylserine phospholipid is DSPC, DMPS, or a mixture thereof.
  • the pH value of the diluting solution is about 12.0 ⁇ 3 0, 12.0 ⁇ 2.0, 11.0 ⁇ 1.5, 11.0 ⁇ 1.0 , 11.0 ⁇ 0.9, 11.0 ⁇ 0.8 , 11.0 ⁇ 0.7, 11.0 ⁇ 0.6, 11.0 ⁇ 0 5. 1100 ⁇ 0.4, 11.0 ⁇ 0.3, 11.0 ⁇ 0.2, or 11.0 ⁇ 0.1 (e.g., about 11.6).
  • the pH value of diluting solution is about 5.0 ⁇ -2.0 , 5.0 ⁇ 1.5, 5.0 ⁇ 1.0, 5.0 ⁇ 0.9, 5.0 ⁇ 0.8, 5.0 ⁇ 0.7, 5.0 ⁇ 0.6, 5.0 ⁇ 0.5, 5.0 ⁇ 0.4, 5.0 ⁇ 0.3, 5.0 ⁇ 0.2, or 5.0 ⁇ 0.1 (e.g., about 4.4).
  • Phospholipids may assemble into one or more lipid bilayers.
  • phospholipids comprise a. phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid, moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g., delivery of one or more elements to a. target tissue.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g., delivery of one or more elements to a. target tissue.
  • elements e.g., a therapeutic agent
  • LNPs
  • Particular phospholipids can facilitate cellular uptake and/or fusion.
  • an anionic phospholipid e.g., phospholipids comprising phosphatidyl glycerol or phosphatidylserine
  • a cell e.g. , receptors of a cellular or intracellular membrane.
  • Cellular uptake of a lipid-containing composition e.g, LNPs
  • Anionic phospholipids can also facilitate fusion to a membrane.
  • Interaction of an anionic phospholipid with a receptor of a cell can bring elements of a lipid-containing composition into contact with a membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g, a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g, delivery of one or more elements to a target tissue.
  • a membrane e.g, a cellular or intracellular membrane.
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g, an alkenyl group in which one or more double bonds are replaced with a triple bond).
  • an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a. useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidyl glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipids, such as sphingomyelin.
  • a therapeutic and/or prophylactic is a protein, for example, a protein needed to augment or replace a naturally occurring protein of interest.
  • proteins or polypeptides may be naturally occurring, or may be modified using methods known in the art, e.g., to increase half-life.
  • Exemplary proteins are intracellular, transmembrane, or secreted.
  • a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) of the disclosure further includes one or more ionizable lipids in addition to the sialic acid lipid of Formula (SA-I).
  • the ionizable lipid is of compound of Formula (IL-A): (IL-A) or its N -oxide, or a salt or isomer thereof, wherein:
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R 2 and R J are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*0R”, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of hydrogen, a C 3-6 carbocycle, -(CH 2 ) n Q, and unsubstituted C 1- 6 alkyl, where Q is selected from a carbocycle, heterocycle, and and , wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S; and a is
  • each o is independently selected from 1, 2, 3, and 4 and each n is independently selected from 1, 2, 3, 4, and 5;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , Ci-6 alkyl, -OR, -S(O) 2 R, - S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • R 12 is selected from the group consisting of H, OH, C 1-3 alkyl, and C 2-3 alkenyl; each R is independently selected from the group consisting of C 1-6 alkyl, C 1-3 alkyl-aryl, C 2-3 alkenyl, and H;
  • R A is selected from H and C 1-3 alkyl
  • R SX is selected from a C 3-8 carbocycle, a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, C 1-6 alkyl, C 2-6 alkenyl, (C 1-3 alkoxy )C 1-3 alkyl, (CH 2 ) p1 O(CH 2 ) P2 R SX1 , and (CH 2 ) p1 R SX1 , wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C 1-6 alkyl, and (C 1-3 alkoxy)C 1-3 alkyl;
  • R SX1 is selected from C(O)NR 14 R 14 ’, a C 3-8 carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C 1-3 alkyl, (C 1-3 alkoxy )C 1-3 alkyl, C 1-6 alkylamino, di-(C 1-6 alkyl) amino, and NH 2 ; each R 13 is selected from the group consisting of OH, oxo, halo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 1-6 alkylamino, di-(C 1-6 alkyl) amino, NH 2 , C(O)NH 2 , CN, and NO 2 ;
  • R 14 and R’ 4 are each independently selected from the group consisting of H and C 1-6 alkyl; p1 is selected from 1, 2, 3, 4, and 5; p 2 is selected from 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H; each R b is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H; R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M’ are independently selected from an aryl group, and a heteroaryl group, in which M” is a bond, C 1-13 alkyl or C 2- 1 3 alkenyl; each R M is independently selected from the group consisting of H, C 1-6 alkyl and
  • each R’ is independently selected from the group consisting of C 1 -18 alkyl, C 2-18 alkenyl.
  • each R” is independently selected from the group consisting of C 3-15 alkyl and C3-15 alkenyl;
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 1 1 , 12, and 13.
  • the ionizable lipid is of compound of Formula (IL-B): (IL-B) or its N-oxide, or a. salt or isomer thereof, wherein R’ a is R branched. branched - s. ; wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ , R a ⁇ , andR a ⁇ are each independently selected from the group consisting of H, C 2-12 alkyl, and C 2-12 alkenyl;
  • R 2 and R J are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl;
  • R 4 is selected from the group consisting of , wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and wherein denotes a point of attachment;
  • R 10 is N(R) 2 , each R is independently selected from the group consisting of C 1 - 6 alkyl, C 2-3 alkenyl, and H; and n 2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R 5 is independently selected from the group consisting of C 1-3 alkyl,
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl,
  • M: and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
  • R’ is a C 1-12 alkyl or C 2-12 alkenyl
  • 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • the ionizable lipid is of compound a compound of Formula (IL- C): a salt or isomer thereof, wherein
  • 1 is selected from 1, 2, 3, 4, and 5;
  • M 1 is M' ;
  • R 4 is -(CH 2 ) n Q, in which Q is OH, and n is selected from 1, 2, 3, 4, or 5;
  • M and M’ are independently selected from -C(O)O- and -OC(O)-;
  • R 2 and R 3 are both C 1 -14 alkyl, or C 2-14 alkenyl
  • R’ is a C 1 -C 12 linear alkvl.
  • the ionizable lipid is of compound a compound of Formula (II
  • R 2 and R J are each independently selected from the group consisting of C 1-14 alkyl and C2-14 alkenyl;
  • R 4 is wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;
  • R’ is a C 1-12 alkyl or C 2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable lipid is a. of compound of Formula. (IL-I):
  • R 1 is selected from the group consisting of C 5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1 -14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of hydrogen, a C 3-6 carbocycle, , and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, , each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; each R 5 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H; each R 6 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M’ are independently selected from ( ) ( ) ( ) ( ) S(O) 2 -, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C 1 -13 alkyl or C 2- 13 alkenyl;
  • R 7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
  • R 8 is selected, from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, -OR, -S(O) 2 R, - S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • R 10 is selected from the group consisting of H, OH, C 1-3 alkyl, and C 2-3 alkenyl; each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, (CH 2 ) q OR*, and H, and each q is independently selected from 1, 2, and 3; each R’ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C 3-15 alkyl and
  • each R* is independently selected from the group consisting of C 1-12 alkyl and
  • the ionizable lipid is a of compound, of Formula (IL-IA): or a salt or isomer thereof, wherein 1 is selected from 1 , 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;
  • M 1 is a bond or M’
  • R 4 is un substituted C 1-3 alkyl, or -(CH 2 ) n Q, in which Q is or heteroaryl, and each n is selected from 1, 2, 3, 4, or 5;
  • M and M’ are independently selected from , art aryl group, and a heteroaryl group
  • R 2 and R 3 are both C 1-14 alkyl or C 2-14 alkenyl
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; and
  • R’ is a C 1 -20 alkyl or C 2-18 alkenyl.
  • the ionizable lipid is a. compound of Formula (IL-IB): (IL-IB), or its N-oxide, or a salt or isomer thereof, wherein
  • R is selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl
  • R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, and C 2- 14 alkenyl;
  • M and M’ are independently selected from -C(O)O- and -OC(O)-;
  • R N is H, or C 1-3 alkyl
  • X a and X b are each independently O or S;
  • R 10 is selected from the group consisting of H, halo, heteroaryl, a carbocycle, a. heterocycle, aryl and heteroaryl; each R is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; r is 0 or 1; t 1 is selected from 1, 2, 3, 4, and 5; p 1 is selected from 1, 2, 3, 4, and 5; q 1 is selected from 1, 2, 3, 4, and 5; and s 1 is selected from 1, 2, 3, 4.
  • the ionizable lipid is a compound of Formula (IL-IC): (IL-IC), or its N-oxide, or a salt or isomer thereof, wherein R’ a is or ; wherein is and is: ; and
  • R ,b is: wherein denotes a point of attachment; wherein R a ⁇ , R a ⁇ and R ay are each C 1-12 alkyl or C 2-12 alkenyl;
  • R b ⁇ is H, C 1 -12 alkyl or C 2-12 alkenyl
  • R 2 and R 3 are each independently selected from the group consisting of C 1-14 alkyl and C 2-14 alkenyl;
  • R 4 is -(CH 2 ) n OH; or wherein denotes a point of attachment; each R’ independently is a C 1-12 alkyl or C 2-12 alkenyl;
  • R 10 is N(R) 2 ; each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; and n and n2 are each selected from the group consisting of 1 , 2, 3, 4, and 5;
  • Y a is a C 3-6 carbocycle
  • R*” a is selected from the group consisting of C 1-15 alkyl and C 2-15 alkenyl
  • the ionizable lipid is a compound of Formula (IL*) or a salt thereof, wherein:
  • R 1 is -OH, -NR N -C 4-10 cycloalkenyl optionally substituted with one or more oxo or
  • R N IS H or C 1-6 alkyl
  • R is H or C 1-6 alkyl
  • R N ’ is H or C 1-6 alkyl, o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8, m is 4, 5, 6, 7, or 8;
  • M is - wherein * indicates attachment to R 2 ;
  • M’ is wherein * indicates attachment to R 3 ;
  • R 2 is or -(C 1-6 alkylene)-(C 3-8 cycloalkyl)-C 1-6 alkyl;
  • R 2a is -H or C 1-10 alkyl
  • R 2b is -H or C 1-10 alkyl
  • R 2C is C 1-8 alkyl or C 2-8 alkenyl; R 3 is
  • R 3a is H or C 1-10 alkyl
  • R 3b is H ar C 1-8 alkyl
  • R to is C 1-10 alkyl or C 2-8 alkenyl.
  • the ionizable lipid is of Formula (IL**-I): or a salt thereof, wherein :
  • R 1 is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8;
  • R 2C is C 4-8 alkyl
  • R 3a is C 7-10 alkyl
  • R 3C is C 3-5 alkyl.
  • the ionizable lipid is of Formula (IL**-III): or a salt thereof, wherein : R 1 is NR N - C 4-10 cycloalkenyl optionally substituted with one or more oxo or -
  • R N is H
  • R is C 1-2 alkyl
  • R N is H, o is 2, 3, or 4; n is 6, 7, or 8;
  • R 2a is C 7-10 alkyl
  • R 2C is C 4-6 alkyl
  • R 3a is C 1-3 alkyl
  • R to is C 4-6 alkyl.
  • the ionizable lipid is of Formula (IL**-IV): or a salt thereof, wherein:
  • R 1 is OH; o is 2, 3, or 4; n is 6, 7, or 8;
  • R 2b is C 3-5 alkyl
  • R 2C is C 2-4 alkyl
  • R 3a is C 7-10 alkyl
  • R to is C 4-6 alkyl.
  • the ionizable lipid is of Formula (IL*-I); or a salt thereof, wherein:
  • R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*;
  • R 3a is C 1-8 alkyl.
  • ionizable lipid is of Formula (IL*-Ia): or a salt thereof, wherein:
  • R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*;
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula. (IL*-Ia’): or a salt thereof, wherein: o, , and R 3c are as defined for variable IL*, and
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula. (IL *-IIa): or a salt, thereof, wherein:
  • R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for Formula IL*; and R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-II'): or a salt thereof, wherein: o, M, XL, R 2C and R 3c are as defined for variable IL*; and
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-III): or a salt thereof, wherein:
  • R 3 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*;
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein:
  • R 3 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*;
  • R 2b is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein:
  • R 1 , o, M, M’, R 2C , and R 3c are as defined for variable IL*;
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIa’): or a salt thereof, wherein:
  • R 1 , o, M, M ⁇ R 2C , and R 3c are as defined for variable IL*;
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (ILMIIb): or a salt thereof, wherein;
  • R 1 , o, M, M', R 2c , and R 3c are as defined for variable IL*,
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (IL*-IIIb’): or a salt thereof, wherein;
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • the ionizable lipid is of Formula. (IL*-IV): or a salt thereof, wherein;
  • R 1 , o, m, n, M, M’, R 2c and R 3c are as defined for variable IL*;
  • R 3a is C 1-8 alkyl.
  • R 1 , o, m, n, M, M’, R 2c , and R 3c are as defined for variable IL*;
  • R 2b is a C 1-8 alkyl
  • R 3 a is C 1-8 alkyl.
  • the ionizable lipid is of Formula (JL*-Iva’): or a salt thereof, wherein: o, M, M’, R 2c , and R 3c are as defined for variable IL*,
  • R 2a is a C 1-8 alkyl
  • R 3a is C 1-8 alkyl.
  • o is 1.
  • o is 2.
  • o is 3.
  • o is 4.
  • R 1 is -OH.
  • R N is H.
  • R N is methyl
  • R N is ethyl
  • R 1 is -NR N -cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(R N ’R N ’).
  • R N is H.
  • R N is methyl
  • R N ’ is ethyl
  • R ⁇ is II
  • R N is methyl
  • R N is ethyl
  • R N is H and R N is methyl.
  • R 1 is [0337] In some embodiments of the ionizable lipid, R 1 is [0338] In some embodiments of the ionizable lipid. R 1 is
  • m is 4.
  • m is 5.
  • m is 6.
  • m 7.
  • m 8.
  • m is 4.
  • n is 5.
  • n is 6.
  • n 7.
  • n 8.
  • n 5 and m is 7.
  • n 7 and m is 7.
  • m is 6 and n is 6.
  • M is wherein * indicates attachment to R 2 .
  • M is wherein * indicates attachment to R 2 .
  • M’ is wherein * indicates attachment to R 3 .
  • M’ is wherein * indicates attachment to R 3 .
  • M is wherein * indicates attachment to R 2
  • M’ is wherein * indicates attachment to R 3
  • R 2 is
  • R 2a is hydrogen
  • R 2a is methyl
  • R 2a is ethyl
  • R 2a is propyl
  • R 2a is butyl
  • R 2a is pentyl
  • R 2a is hexyl
  • R 2a is heptyl
  • R 2a is octyl
  • R 2b is hydrogen
  • R 2b is methyl
  • R 2b is ethyl
  • R 2b is propyl
  • R 2b is butyl
  • R 2b is pentyl
  • R 2b is hexyl
  • R 2b is heptyl
  • R 2b is octyl
  • R 2a is hydrogen and R 2b is hydrogen
  • R 2a is hexyl and R 2b is hydrogen.
  • R 2a is octyl and R 2b is hydrogen.
  • R 2a is hydrogen and R 2b is butyl.
  • R 2c is methyl
  • R 2c is ethyl
  • R 2c is propyl
  • R 2c is butyl
  • R 2c is pentyl
  • R 2c is hexyl
  • R 2c is heptyl
  • R 2c is octyl.
  • R 2 is --( C 1-6 alkylene)-(C 3-8 cycloalkyl)- C 1-6 alkyl.
  • R 2 is --( C 1-6 alkylene )-(cyclohexyl)-C 1-6 alkyl.
  • R 2 is -(C 1-6 alkylene)-(cyclopentyl)-C 1-6 alkyl.
  • R 3 is
  • R 3a is hydrogen
  • R 3a is methyl
  • R 3a is ethyl
  • R 3a is propyl
  • R 3a is butyl
  • R 3a is pentyl
  • R 3a is hexyl
  • R 3a is heptyl
  • R 3a is octyl
  • R 3b is hydrogen
  • R 3b is methyl
  • R 3b is ethyl
  • R 3b is propyl
  • R 3b is butyl
  • R 3b is pentyl
  • R 3b is hexyl
  • R 3b is heptyl
  • R 3b is octyl
  • R 3a is octyl and R 3b is hydrogen.
  • R 3a is ethyl and R 3b is hydrogen.
  • R 3a is hexyl and R 3b is hydrogen.
  • R 3c is methyl
  • R 3c is ethyl.
  • R 3c is propyl.
  • R 3c is butyl
  • R 3c is pentyl
  • R 3c is hexyl
  • R 3c is heptyl
  • R 3c is octyl
  • variables o, R 1 , R N , R N , R N , m, n, M, M’, R 2 , R 2a , R 2b , R 2C , R ’, R 3a , R 3b , and R 3c can each be, where applicable, selected from the groups described herein, and any group described herein for any of variables O..R 1 , R N , R N , R N ⁇ m, n, M, M’, R 2 , R 2a , R 2b , R 2C , R 3 , R 3a , R 3b .
  • 3c can be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R 1 , R N , R N ’, R N , m, n, M, M’, R 2 , R 2a , R 2b , R 2C , R 3 , R 3a , R 3b , and R 3c .
  • the ionizable lipid is a compound selected from Table IL-1.
  • the ionizable lipid is a compound selected from Table IL-2.
  • the ionizable lipid is a compound of Formula (IL-IIA): (IL-IIA), or its N-oxide, or a salt or isomer thereof, wherein: m is selected from 5, 6, 7, 8, and 9;
  • R 2 and R 3 are each independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl;
  • R 4 is selected from -(CH 2 ) n OH, wherein n is selected from 1, 2, 3, 4, and 5, and wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R 10 is -
  • each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H;
  • M is selected from -OC(O)O-, -C(O)O ⁇ , -O-M”-O-, and -N(R M )C(O)-, in which M” is -(CH 2 ) Z C(O)-, wherein z is 1, 2, 3, or 4;
  • M is -(CH 2 ) z C(O>, C 1-13 alkyl, -B(R**), or -Si(R**) 2 -; z is 1, 2, 3, or 4, each R M is independently selected from H and C 1-6 alkyl; each R** is independently selected from H and C 1-12 alkyl;
  • R’ a is C 1- 18 alkyl, C2-18 alkenyl, or -R*YR*”, wherein: each R*” is independently C 1 -15 alkyl; each R* is independently C 1-12 alkyl; each Y is independently a C 3-6 carbocycle; and
  • R is a C 3 -C 13 alkyl, optionally substituted with OH.
  • the ionizable lipid is a. compound of Formula (IL-IIAX): (IL-IIAX) or its N-oxide, or a salt or isomer thereof, wherein:
  • R 1 is -R”M’R’, wherein: each R’ is independently C 1 -18 alkyl;
  • R 2 and R 3 are each independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl;
  • R 4 is selected from -(CH 2 ) n OH, wherein n is selected from 1, 2, 3, 4, and 5, and , wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and.
  • R 10 is - N(R) 2 , wherein each R is independently selected from the group consisting of C 1-6 alkyl, C 2-3 alkenyl, and H; each R 5 is H; each R 6 is H; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.
  • the ionizable lipid is a compound selected from Table IL-3.
  • the ionizable lipid is a compound of Formula (IL-IIB): (IL-IIB) or its N-oxide, or a salt or isomer thereof, wherein R’ a is: and R ’ b is: wherein denotes a point of attachment;
  • R a ⁇ , R a ⁇ , and R a ⁇ are each independently selected from the group consisting of H,
  • R b ⁇ , R b ⁇ and R b ⁇ are each independently selected from the group consisting of H,
  • R b ⁇ , R b ⁇ and R b ⁇ is selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • R 2 and R J are each independently selected from the group consisting of C 1 -14 alkyl and
  • R 4 is selected from -(CH 2 ) n NRTQ, -(CH 2 ) n NRS(O) 2 TQ, -(CH 2 ) n NRC(O)H and
  • n is selected from 1, 2, 3, 4, and 5;
  • T is a bond or a C 1 -3 alkyl linker, C 2-3 alkenyl linker, or C 2-3 alkynyl linker;
  • Q is selected from 3-14 membered heterocycle containing 1-5 heteroatoms selected from N, O, and S, C 3-10 carbocycle, C 1-6 alkyl, and C 2-6 alkenyl, wherein the alkyl, alkenyl, heterocycle, and carbocycle are each optionally substituted with one or more R Q ;
  • each R Q independently is selected from the group consisting of oxo, hydroxyl, cyano, amino, C 1-6 alkylamino, di-C 1-6 alkylamino, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 1-6 alkanolyl, -C(O)C 1-6 alkyl, and -NRC(O) C 1-6 alkyl;
  • each R is independently selected from H, C 1-6 alkyl, and C 2-6 alken
  • 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
  • the ionizable lipid is a compound selected from Table IL-4.
  • the ionizable lipid is a compound selected from Table IL-5.
  • the ionizable lipid is a. compound of Formula (IL-IIC):
  • IL-IIC IL-IIC
  • R aa and R ap are each independently selected from the group consisting of H and C 1-2 alkyl, wherein at least one of R aa and R* 3 is a C 1 or C 2 alkyl;
  • R’ is selected from the group consisting of C 1 -18 alkyl and C 2-18 alkenyl
  • R 2 and R 3 are each independently selected from the group consisting of C 1 -14 alkyl and C 2-14 alkenyl;
  • R 4 is -(CH 2 ) n Q, wherein n is independently selected from 1, 2, 3, 4, and. 5, where Q is selected from NRS(O) 2 R sx and , wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S; and a is 1, 2, 3, or
  • R is selected from H and C 1 -3 alkyl
  • R sX is selected from a C 3-8 carbocycle, a 3-14 membered, heterocycle containing one or more heteroatoms selected from N, O and S, C 1-6 alkyl, C 2-6 alkenyl, (C 1-3 alkoxy)C 1-3 alkyl, and wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C 1-6 alkyl, and (C 1-3 alkoxy)C 1-3 alkyl;
  • R SX 1 is selected from C(O)NR 14 R 14 ’, a C 3-8 carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C 1-3 alkyl, (C 1-3 alkoxy )C 1-3 alkyl, C 1-6 alkylamino, di-(C 1-6 alkyl) amino, and NH 2 ; each R k ’ is selected from the group consisting of OH, oxo, halo, C 1-6 alkyl, C 1-6 alkoxy, C 2-6 alkenyl, C 1-6 alkylamino, di-(C 1-6 alkyl) amino, NH 2 , C(O)NH 2 , CM, and NO 2 ;
  • R 14 and R 14 are each independently selected from the group consisting of H and C 1-6 alkyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
  • p 1 is selected, from 1, 2, 3, 4, 5, 6, 7, 8, and 9; p 1 is selected from 1, 2, 3, 4, and 5; and
  • P 2 is selected from 1, 2, 3, 4, and 5.
  • the ionizable lipid is a. compound selected from Table IL-6.
  • the ionizable lipid is a compound of Formula (IL-III): (IL-III), or salts or isomers thereof, wherein,
  • W is ring A is t is 1 or 2;
  • a 1 and A 2 are each independently selected, from CH or N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1 ) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, -R” MR', -R*YR”, -YR”, and -R*OR”;
  • R x1 and R x2 are each independently H or C 1-3 alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, - OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, - P(O)(OR’)O-, -S(O) 2 -, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;
  • M* is C 1 -C 6 alkyl
  • W 1 and W 2 are each independently selected from the group consisting of -O- and - N(R 6 )-; each R 6 is independently selected from the group consisting of H and C 1-5 alkyl;
  • X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, -CH 2 -, -(CH 2 ) 2 -, -CHR-, -CHY-, -C(O)-, -C(O)O ⁇ , -OC(O)-, -(CH 2 )n-C(O)-, -C(O)-(CH 2 ) n -, -(CH 2 ) tl - C(O)O-, -OC(O)-(CH 2 )n-, -(CH 2 )n-OC(O)-, -C(O)O-(CH 2 )n-, -CH(OH)-, -C(S)-, and - CH(SH)-; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; each R is independently selected from the group consisting
  • the ionizable lipid is a compound of Formula (IL-IIIA): or a. salt or isomer thereof, wherein R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, -R”MR’, -R*YR", -YR”, and -R*OR”; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O ⁇ , -C(O)N(R’) ⁇ , -N(R’)C(O)-, -C(O>, -C(S)-, -C(S)S-, -SC(S)
  • X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, -CH 2 -, -(CH 2 ) 2 -. -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH 2 -, -CH 2 -C(O)-, -C(O)O-CH 2 -, -OC(O)-CH 2 -, -CH 2 -C(O)O-, -CH 2 -OC(O)-, -CH(OH)-, -C(S) ⁇ , and -CH(SH)-; each Y is independently a C 3-6 carbocycle; each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl; each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-5 carbocycle; each R’ is independently
  • the ionizable lipid is a compound selected from Table IL-7.
  • the ionizable lipid is a compound selected from:
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the ionizable lipid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • an ionizable lipid may have a positive or partial positive charge at physiological pH.
  • Such lipids may be referred to as cationic or ionizable (amino)lipids.
  • Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and. a negative charge.
  • PEG lipid refers 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.
  • PEGylated lipids PEGylated lipids.
  • a PEG- lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG lipid includes, but not limited to, 1,2-dimyristoyl-sn- glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxipropyl-3-amine (PEG-c-DMA) .
  • PEG-DMG 1,2-dimyristoyl-sn- glycerol methoxy polyethylene glycol
  • PEG-DSPE 1,2-distearoyl-s
  • the PEG lipid is selected from the group consisting of a PEG- modified phosphatidylethanol amine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof
  • the lipid moiety of the PEG lipids includes those having lengths of from about. C 14 to about C 22 , preferably from about C 14 to about C 16 .
  • a PEG moiety for example, an mPEG-NH 2 , has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG lipid is PEG2k-DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • PEG lipid which is a non-diffusible PEG.
  • non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • PEG lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
  • the lipid, component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids.
  • a PEG lipid is a lipid modified with polyethylene glycol.
  • a PEG- lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanol amines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified di acyl glycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG-modified lipids are a modified form of PEG DMG.
  • PEG-DMG has the following structure:
  • the PEG-modified lipids are a modified form of PEG-DSG.
  • PEG-DSG has the following structure:
  • PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG- chain.
  • the PEG lipid is a. PEG-OH lipid.
  • a “PEG-OH lipid” (also referred to herein as “hydroxy -PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (- OH) groups on the lipid.
  • the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain.
  • a PEG-OH or hydroxy-PEGylated lipid comprises an OH group at. the terminus of the PEG chain.
  • a PEG lipid useful in the present invention is a compound of Formula (PL-I).
  • PL-I compounds of Formula (PL-I): or salts thereof, wherein:
  • R 3 is OR O
  • R O is hydrogen, optionally substituted alkyl, or an oxygen-protecting group; r is an integer between 1 and 100, inclusive;
  • L 1 is optionally substituted C 1 -10 alkylene, wherein at least one methylene of the optionally substituted C 1 -10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene,
  • D is absent; or D is a moiety obtained by dick chemistry or a moiety cleavable under physiological conditions; m is O, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10;
  • each instance of L 2 is independently a bond or optionally substituted C 1-6 alkylene, wherein one methylene unit of the optionally substituted C 1-6 alkylene is optionally replaced with each instance of R 2 is independently optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally wherein one or more methylene units of R 2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen-protecting group;
  • Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.
  • the compound of Formula (PL-I) is a PEG-OH lipid (i.e., R 3 is -OR O , and R O is hydrogen). In some embodiments, the compound of Formula (PL-I) is of Formula (PL-I-OH):
  • a PEG lipid useful in the present invention is a PEGylated fatty acid.
  • a PEG lipid useful in the present invention is a compound of Formula (PL-11).
  • R 3 is--OR O ;
  • R O is hydrogen, optionally substituted alkyl or an oxygen -protecting group, r is an integer between 1 and 100, inclusive;
  • R 5 is optionally substituted C10-40 alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40 alkynyl; and optionally one or more methylene groups of R' are replaced with optionally substituted, carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, , and each instance of R N is independently hydrogen, optionally substituted alkyl, or a nitrogen-protecting group .
  • the compound of Formula (PL-II) is of Formula (PL-II-OH): (PL-II-OH), or a salt thereof.
  • r is 35-55. In some embodiments, r is 45.
  • the compound of Formula. (PL-II) is: (PL-01), or a salt thereof.
  • r is 1 -100. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45.
  • the compound of Formula (PL-II) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2
  • the PEG lipid is PEG1.
  • PEG1 is a plurality of compounds of Formula (PL-01): or salts thereof, wherein r is 1-100. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45.
  • the PEG lipids may be one or more of the PEG lipids described, in U.S. Application No. 62/520,530.
  • structural lipid refers to sterols and also to lipids containing sterol moieties.
  • Structural lipids can be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof
  • the structural lipid is a sterol.
  • “sterols” are a subgroup of steroids consisting of steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha-tocopherol.
  • the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62/520,530.
  • the lipid nanoparticles and pharmaceutical compositions comprising same of the disclosure can be used to deliver therapeutic and/or prophylactic agents to a target cell, tissue, organ or subject.
  • the therapeutic agent can be a polynucleotide, such as mRNA or DNA.
  • the polynucleotide can encode a polypeptide, such as a therapeutic agent or therapeutic polypeptide, or a CRISPR protein for use in gene editing.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to human CD33+ hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to CD169+ marginal Macrophages.
  • LNPs comprising Compound No. 9 are suitable for delivery of nucleic acids encoding polypeptides to promote an immune response (e.g., any antigen described herein). In some embodiments, the LNPs improve the quality of the immune response.
  • LNPs comprising Compound No. 1 are for delivery of nucleic acids encoding polypeptides for therapeutic use when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 1 show reduced immune response compared to LNPs that do not comprise Compound No. 1.
  • LNPs comprising Compound No. 9 are for delivery of nucleic acids encoding polypeptides for therapeutic use when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 9 show reduced immune response compared to LNPs that do not comprise Compound No. 9.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
  • myeloid progenitor cells e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of a. nucleic acid encoding a. therapeutic polypeptide to hematopoietic stem and progenitor cells, e.g. HCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
  • a. nucleic acid encoding a. therapeutic polypeptide to hematopoietic stem and progenitor cells, e.g. HCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
  • LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to erythroid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a. CRISPR protein and/or gRNA to erythroid progenitor cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acid encoding CRISPR protein and/or gRNA to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
  • LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to hematopoietic stem and. progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38-HSPCs.
  • the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates or produces de novo) protein expression.
  • agents that enhances include RNAs, mRNAs, dsRNAs, CRISPR/Cas based systems, as well as single and double stranded DNAs (e.g., expression vectors).
  • therapeutic agent comprises an mRNA.
  • the agent that upregulates protein expression may upregulate expression of a naturally occurring or non- naturally occurring protein (e.g., a chimeric protein that has been modified to improve half- life, a fusion protein, or one that comprises desirable amino acid changes).
  • exemplary proteins include intracellular, transmembrane, or secreted proteins, gene editing proteins, peptides, or polypeptides.
  • the mRNA may encode the protein whose expression is upregulated, and upon delivery to the target cell, tissue, organ or subject, be translated to produced increased expression of the protein.
  • the mRNA may encode a protein that indirectly upregulates the expression of one or more proteins.
  • the mRNA may encode a natural or synthetic transcriptional activator.
  • the therapeutic agent comprises a DNA therapeutic agent (e.g., a DNA molecule).
  • the DNA molecule can be a double-stranded DNA, a single-stranded DNA. (ssDNA), or a molecule that is a partially double-stranded DNA, i.e., has a portion that is double-stranded and a portion that is single-stranded.
  • the DNA molecule is triple-stranded or is partially triple-stranded, i.e., has a portion that is triple stranded and a portion that is double stranded.
  • the DNA molecule can be a circular DNA molecule or a linear DNA molecule.
  • a DNA therapeutic and/or prophylactic agent can be a DNA molecule that is capable of transferring a. gene into a cell, e.g., that encodes and can express a. transcript.
  • the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule produced in vitro.
  • the DNA molecule is a recombinant molecule.
  • Non- limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.
  • the DNA therapeutic and/or prophylactic agents described herein, e.g., DNA vectors can include a variety of different features.
  • the DNA therapeutic and/or prophylactic agents described herein, e.g., DNA vectors can include a non-coding DNA sequence.
  • a DNA sequence can include at least one regulatory element for a. gene, e.g., a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, 5’ untranslated region (UTR), 3’ UTR and the like.
  • the non-coding DNA sequence is an intron.
  • the non-coding DNA sequence comprises a pair of inverted terminal repeats (ITRs), e.g.
  • a DNA sequence described herein can have a non-coding DNA sequence that is operatively linked to a gene that is transcriptionally active.
  • a DNA sequence described herein can have a non-coding DNA sequence that is not linked to a gene, i.e., the non-coding DNA does not regulate a gene on the DNA. sequence.
  • the one or more therapeutic and/or prophylactic agents are selected from the group consisting of a plasmid expression vector, a viral expression vector, and mixtures thereof
  • the RNA when the therapeutic and/or prophylactic agents comprise an RNA, the RNA is selected from the group consisting of a single-stranded RNA, a double-stranded RNA (dsRNA), a partially double-stranded RNA, and mixtures thereof.
  • the RNA is selected from the group consisting of a circular RNA, a linear RNA, and mixtures thereof.
  • the RNA when the therapeutic and/or prophylactic agents comprise an RNA, the RNA is selected from the group consisting of a spliceosomal RNA, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), an RNA. interference (RNAi) molecule, a microRNA (miRNA ), an antagomir, an antisense RNA, a ribozyme, a Dicer substrate RNA (dsRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), locked nucleic acids (LNAs) and a guide RNA for a CRISPR/Cas system, and mixtures thereof.
  • a spliceosomal RNA a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), an RNA. interference (RNAi) molecule, a microRNA (miRNA ), an antagomir, an antisense RNA,
  • the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA. (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA a small interfering RNA
  • miRNA asymmetrical interfering RNA.
  • miRNA microRNA
  • dsRNA Dicer substrate RNA
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • the therapeutic and/or prophylactic agents comprise RNA and DNA, e.g. as an RNA-DNA hybrid.
  • the one or more therapeutic and/or prophylactic agents comprise an mRNA.
  • the one or more therapeutic and/or prophylactic agents comprise a modified mRNA (mmRN.A).
  • the mRNA comprises Nl- methyl-pseudouridine.
  • the mRNA comprises nucleosides selected from the group consisting of N1-methyl-pseudouridine, adenosine, guanosine, and cytidine, for example in the open reading frame of the mRNA.
  • the one or more therapeutic and/or prophylactic agents comprise an mRNA that incorporates a micro-RNA binding site (miR binding site).
  • an mRNA includes one or more of a stem loop, a chain-terminating nucleoside, a poly A. sequence, a poly adenylation signal, and/or a 5' cap structure.
  • An mRNA may be a naturally or non-naturally occurring mRNA.
  • An mRNA may include one or more modified nudeobases, nucleosides, or nucleotides, as described below, in which case it may be referred to as a “modified mRNA” or “mmRNA.”
  • nucleoside is defined as a compound containing a sugar molecule (e.g, a pentose or ribose) or derivative thereof in combination with an organic base (e.g, a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • nucleotide is defined as a nucleoside including a. phosphate group.
  • An mRNA may include a 5' untranslated region (5'-UTR), a 3' untranslated region (5'- UTR), and/or a coding region (e.g., an open reading frame).
  • An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000) of base pairs.
  • nudeobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring.
  • all of a particular nucleobase type may be modified.
  • all uracils or uridines are modified.
  • the mRNA can be referred to as “fully modified,” e.g, for uracil or uridine.
  • an mRNA may include a 5' cap structure, a chain-terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and/or a polyadenylation signal.
  • a Kozak sequence also known as a Kozak consensus sequence
  • a 5' cap structure or cap species is a compound including two nucleoside moi eties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA).
  • a cap species may include one or more modified nucleosides and/or linker moieties.
  • a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG.
  • a cap species may also be an anti-reverse cap analog.
  • a non-limiting list of possible cap species includes
  • An mRNA may instead or additionally include a chain-terminating nucleoside.
  • a chain-terminating nucleoside may include those nucleosides deoxygenated at the 2' and/or 3' positions of their sugar group.
  • Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxy cytosine, 3' deoxyguanosine, 3' deoxythymine, and 2 ',3' dideoxynucleosides, such aass 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2',3' dideoxyguanosine, and 2',3' dideoxythymine.
  • incorporation of a chain-terminating nucleotide into an mRNA for example at the 3 '-terminus, may result in stabilization of the mRNA.
  • An mRNA may instead or additionally include a stem loop, such as a histone 3’ UTR stem loop.
  • a stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs.
  • a. stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs.
  • a stem loop may be located in any region of an mRNA.
  • a stem loop may be located in, before, or after an untranslated region (a 5' untranslated region or a 3' untranslated region), a coding region, or a poly A sequence or tail.
  • a stem loop may affect one or more function(s) of an mRNA, such as nucleocytoplasmic transport, stability, initiation of translation, translation efficiency, and/or transcriptional termination.
  • An mRNA may instead or additionally include a poly A sequence and/or polyadenylation signal.
  • a polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof.
  • a poly A sequence may also comprise stabilizing nucleotides or analogs.
  • a. poly A sequence can include deoxythymidine, e.g, inverted (or reverse linkage) deoxythymidine (d.T), as a stabilizing nucleotide or analog. Details on using inverted dT and other stabilizing poly A sequence modifications can be found, for example, in WO2017/049275 A2, the content of which is incorporated herein by reference.
  • a polyA sequence may be a.
  • a polyA sequence may affect the nuclear export, translation, and/or stability of an mRNA.
  • Suitable polyA sequences will be known to persons of ordinary skill in the art and include, without limitation the bovine growth, hormone polyadenylation (bgh-PolyA) sequence, the SV40 polyadenylation sequence and the rabbit beta globin (rbGlob) polyadenylation sequence.
  • An mRNA may comprise one or more post-transcriptional regulatory elements, including, without limitation, a nuclear export element such as Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or constitutive transport, element (CTE).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • CTE constitutive transport, element
  • An mRNA may instead or additionally include a microRNA-binding site.
  • MicroRNA- binding sites can be used to regulate mRNA expression in various tissues or cell types.
  • miR-binding sites are engineered into 3’ UTR sequences of an mRNA to regulate, e.g., enhance degradation of mRNA in cells or tissues expressing the cognate miR. Such regulation is useful to regulate or control “off-target” expression in mRNAs, Le., expression in undesired cells or tissues in vivo. Details on using miR-binding sites can be found, for example, in WO 2017/062513 A2, the content of which is incorporated herein by reference.
  • an mRNA is a bicistronic mRNA comprising a first coding region and a second coding region with an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows for internal translation initiation between the first and second coding regions, or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide (e.g. P2A., E2A, F2A and the like).
  • IRES sequences and 2A. peptides are typically used express multiple proteins from the same vector.
  • IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.
  • an mRNA of the disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides (sometimes termed herein “modified mRNAs” or “mmRNAs”).
  • modified mRNAs may have useful properties, including enhanced stability, intracellular retention, enhanced translation, and/or the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, as compared to a reference unmodified mRNA. Therefore, use of modified mRNAs may enhance the efficiency of protein production, intracellular retention of nucleic acids, as well as possess reduced immunogenicity.
  • an mRNA includes one or more (e.g., 1, 2, 3 or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may have reduced degradation in a cell into which the modified mRNA is introduced, relative to a corresponding unmodified. mRNA. [0502] In some embodiments, the modified nucleobase is a modified uracil.
  • nucleobases and nucleosides having a modified uracil include pseudouridine ( ⁇ ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g, 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethyl-uridine (mcm5U), 1 -carboxymethyl
  • -O-methyl-uridine mcm5Um
  • 5-carbamoylmethyl-2'-O-methyl-uridine ncmSUm
  • 5- carboxymethylaminomethyl-2'-O-methyl -uridine cmnmSUm
  • 3,2'-O-dimethyl-uridine m3Um
  • 5-(isopentenylaminomethyl)-2'-O-methyl-uridine inm5Um
  • 1-thio-uridine deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.
  • the modified nucleobase is a modified cytosine.
  • exemplary' nucleobases and nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl -cytidine (ac4C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5- iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1 -methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine
  • the modified nucleobase is a modified adenine.
  • exemplary nucleobases and nucleosides having a. modified adenine include a-thio-adenosine, 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g, 6-chloro-purine), 2-amino ⁇ 6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl- adenine (m1A), 2-methyl
  • the modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include a-thio-guanosine, inosine (I), 1-methyl-inosine (m11), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (oQ), galactosyl -queuosine (galQ), mannosyl -queuosine (tnanQ), 7-cyano-7- deaza-guanosine (preQ
  • a modified mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (c.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified nucleobase is pseudouridine ( ⁇ ), N1- methylpseudouridine (m1 ⁇ ), 2-thiouridine, 4’ -thiouridine, 5-methylcytosine, 2-thio-1-methyl - 1 -deaza- pseudouridine, 2-thio-l -methyl -pseudouridine, 2-thio-5-aza-uridine , 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methyl -pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyl uridine.
  • an mRNA. of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified nucleobase is N1- methylpseudouridine (m1 ⁇ ) and the mRNA of the disclosure is fully modified with N1- methylpseudouridine (m1 ⁇ ).
  • N1 -methylpseudouridine (m1 ⁇ ) represents from 75-100% of the uracils in the mRNA.
  • N1- methylpseudouridine (m1 ⁇ ) represents 100% of the uracils in the mRNA.
  • the modified nucleobase is a modified cytosine.
  • exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2 -thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.
  • a modified mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified nucleobase is a modified adenine.
  • Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1 -methyl- adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).
  • an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (c.g. , a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified nucleobase is a modified, guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methyl -inosine (mil), wyosine (imG), methylwyosine (mim G), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1 ), 7-methyl-guanosine (m7G), 1 -methyl -guanosine (mlG), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.
  • an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified nucleobase is 1-methyl-pseudouridine (mli
  • an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g, a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
  • the modified mRNA comprises pseudouridine ( ⁇ ). In some embodiments, the modified mRNA comprises pseudouridine (y) and 5-methyl-cytidine (rn5C). In some embodiments, the modified mRNA comprises 1-methyl-pseudouridine (m1 ⁇ ). In some embodiments, the modified mRNA comprises 1-methyl-pseudouridine (m1 ⁇ ) and 5-methyl- cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the modified mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the modified mRNA comprises 5-methoxy-uridine (mo5U).
  • the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C).
  • the modified mRNA comprises 2'-O-methyl uridine.
  • the modified mRNA comprises 2'-O-methyl uridine and 5-methyl-cytidine (m5C).
  • the modified mRNA comprises N6-methyl-adenosine (m6A).
  • the modified mRNA comprises N6-methyl-adenosine (m6.A) and 5- methyl-cytidine (m5C).
  • a modified mRNA of the disclosure is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification.
  • a. modified mRNA can be uniformly modified with N1-methylpseudouridine (m1 ⁇ ) or 5-methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with Nl-methylpseudouridine (m1 ⁇ ) or 5-methyl-cytidine (m5C).
  • modified mRNAs of the disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • a modified mRNA of the disclosure may be modified in a coding region (e.g., an open reading frame encoding a polypeptide).
  • a modified mRNA may be modified in regions besides a coding region.
  • a 5'-UTR and/or a 3 -UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications.
  • nucleoside modifications may also be present in the coding region.
  • modified mRNAs of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.
  • nucleoside or nucleotide represents 100 percent of that A, U , G or C nucleotide or nucleoside having been modified. Where percentages are listed, these represent the percentage of that particular A, U, G or C nucleobase triphosphate of the total amount of A, U, G, or C triphosphate present.
  • the combination: 25 % 5-Aminoallyl-CTP + 75 % CTP/ 25 % 5-Methoxy-UTP + 75 % UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of the uracils are UTP.
  • the naturally occurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of those nucleotides found in the polynucleotide. In this example, all of the GTP and ATP nucleotides are left unmodified.
  • the mRNAs of the present disclosure, or regions thereof, may be codon optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove proteins trafficking sequences, remove/add post translation modification sites in encoded proteins (e.g., glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome-binding sites and mRNA degradation sites, adjust translation rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that
  • Codon optimization tools, algorithms and sendees are known in the art; non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA) and/or proprietary methods.
  • the mRNA sequence is optimized using optimization algorithms, e.g., to optimize expression in mammalian cells or enhance mRNA stability.
  • the present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.
  • mRNAs of the present disclosure may be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In some embodiments, mRNAs are made using IVT enzymatic synthesis methods. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.
  • Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis.
  • modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugar.
  • the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain, with chemical synthesis or with a polymerase enzyme.
  • Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.
  • Therapeutic -Agents for Reducing Protein Expression may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.
  • the therapeutic agent is a therapeutic agent that reduces (i.e., decreases, inhibits, or downregulates) protein expression.
  • therapeutic agents that can be used for reducing protein expression include mRNAs that incorporate a micro-RNA binding site(s) (miR binding site), microRNAs (miRNAs), antagomirs, small (short) interfering RNAs (siRNAs) (including shortmers and dicer-substrate RNAs), R'NA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs) and CRISPR/Cas9 technology.
  • miR binding site micro-RNA binding site
  • miRNAs microRNAs
  • antagomirs small (short) interfering RNAs (siRNAs) (including shortmers and dicer-substrate RNAs), R'NA interference (RNAi) molecules, antisense RNAs, ribozymes
  • the prophylactic and/or therapeutic agent comprises a polynucleotide that binds, or encodes a protein that binds, to a target (sensor) sequence present in a target cell, organ, tissue or subject.
  • Sensor sequences include, for example, microRNA (miRNA.) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • miRNA microRNA
  • Non-limiting examples of sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • a polyribonucleotide e.g, a ribonucleic acid. (RNA), e.g, a messenger RNA (mRNA)
  • RNA ribonucleic acid.
  • mRNA messenger RNA
  • ORF open reading frame
  • the sensor sequence comprises a. miRNA-binding site.
  • a miRNA is a 19-25 nucleotide long noncoding RNA that binds to a polyribonucleotide and down -regulates gene expression, either by reducing stability or by inhibiting translation of the polyribonucleotide.
  • a miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA. seed can comprise positions 2-8 or 2- 7 of the mature miRNA.
  • a miRNA seed can comprise 7 nucleotides (e.g, nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA-binding site is flanked by an adenosine (A) opposed to miRNA position 1.
  • a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA- binding site is flanked by an adenosine (A) opposed to miRNA position 1. See, for example, Crimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell.
  • microRNA binding site refers to a sequence within a polyribonucleotide, e.g, within a DNA or within an RNA transcript, including in the 5'UTR and/or 3'UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polyribonucleotide of the disclosure comprising an ORF encoding a. polypeptide further comprises a miRNA-binding site.
  • a 5'UTR and/or 3'UTR of the polyribonucleotide comprises a miRNA-binding site.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA-binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polyribonucleotide, e.g., miRNA-mediated translational repression or degradation of the polyribonucleotide.
  • a miRNA-binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polyribonucleotide, e.g;, miRNA-guided RNA- induced silencing complex (RlSC)-mediated cleavage of mRNA.
  • the miRNA-binding site can have complementarity to, for example, a.
  • a miRNA-binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally occurring miRNA sequence.
  • the desired regulation is mRNA degradation.
  • the miRNA-binding site has full or complete complementarity (e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally occurring miRNA).
  • the mRNA degradation has full or complete complementarity.
  • a miRNA-binding site includes a sequence that has complementarity (e.g, partial or complete complementarity) with a miRNA seed sequence. In some embodiments, the miRNA-binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a. miRNA-binding site includes a sequence that has complementarity partial or complete complementarity) with a miRNA sequence. In some embodiments, the miRNA-binding site includes a sequence that, has complete complementarity with a miRNA sequence. In some embodiments, a miRNA- binding site has complete complementarity with a miRNA sequence, but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA-binding site is the same length as the corresponding miRNA. In some embodiments, the miRNA-binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA-binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3’ terminus, or both. The miRNA-binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA-binding sites or preventing the mRNA from translation.
  • the miRNA-binding site binds to the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA-binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA-binding site or prevents the mRNA from being translated. In some embodiments, the miRNA-binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polyribonucleotide comprising the miRNA-binding site.
  • the miRNA-binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polyribonucleotide comprising the miRNA-binding site.
  • the miRNA-binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polyribonucleotide comprising the miRNA-binding site.
  • the miRNA-binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA-binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about, seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polyribonucleotide By engineering one or more miRNA-binding sites into a polyribonucleotide of the disclosure, the polyribonucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polyribonucleotide.
  • a polyribonucleotide of the disclosure if a polyribonucleotide of the disclosure is not intended to be delivered to a tissue or cell but ends up there, then a. miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5'UTR and/or 3' TR of the polyribonucleotide.
  • miRNA-binding sites can be removed from polyribonucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polyribonucleotide to improve protein expression in tissues or cells containing the miRNA.
  • a polyribonucleotide of the disclosure can include at least one miRNA-binding site in the 5'UTR and/or 3'UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • a polyribonucleotide of the disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5' ⁇ UTR and/or 3'-UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • miRNAs and miRNA-binding sites can correspond to any known sequence, including non-limiting examples described in U.S. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR- 208), endothelial cells (miR- 17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR- 16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR- 1d, miR- 149), kidney (miR- 192, miR- 194, miR-204), and lung epithelial cells (let-7, miR-133, miR- 126).
  • liver miR-122
  • muscle miR-133, miR-206, miR- 208
  • endothelial cells miR- 17-92, miR-126
  • myeloid cells miR-142-3p, miR-142-5p, miR- 16, mi
  • miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g, dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc.
  • APCs antigen-presenting cells
  • Immune cell-specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells-specific miRNAs also regulate many embodiments of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen-presenting cells and displayed on the surface of the antigen-presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
  • binding sites for miRNAs that are known to be expressed, in immune cells, in particular, antigen-presenting cells can be engineered into a polyribonucleotide of the disclosure to suppress the expression of the polyribonucleotide in antigen-presenting cells through miRNA-mediated RNA degradation, subduing the antigen- mediated immune response. Expression of the polyribonucleotide is maintained in non- immune cells where the immune cell-specific miRNAs are not expressed.
  • any miR- 122-binding site can be removed and a.
  • miR-142 (and/or mirR-146)-binding site can be engineered into the 5'UTR and/or 3'UTR of a polyribonucleotide of the disclosure.
  • a polyribonucleotide of the disclosure can include a further negative regulatory; element in the 5'UTR and/or 3'UTR, either alone or in combination with miR-142 and/or miR-146 binding sites.
  • the further negative regulatory element is a Constitutive Decay Element (CDE).
  • Immune cell-specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let- 7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-Iet-7e-5p, hsa-let-7g-3p, hsa-Iet-7g-5p, hsa-let- 7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-1et-7f-l-- 3p, hsa-let-7f-2--5p, hsa-let-7f-5p, miR-125b-l-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR- 130a-5p, miR-132-3p, miR-132-5p, miRNA-130
  • novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:el 18-el27; Vaz C et al., BMC Genomics, 2010, 1 1 ,288, the content of each of which is incorporated herein by reference in its entirety).
  • miRNAs that are known to be expressed in the liver include, but are not limited to, miR- 107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR- 1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR- 199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • miRNA-binding sites from any liver-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the liver.
  • Liver-specific miRNA-binding sites can be engineered, alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs that are known to be expressed in the lung include, but are not limited to, let- 7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a- 3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p.
  • MiRNA- binding sites from any lung-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the lung.
  • Lung-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a- 5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • MiRNA-binding sites from any heart-specific microRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the heart.
  • Heart-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g, APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR- 125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR- 212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p, mi
  • MiRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR- 148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR- 219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • MiRNA-binding sites from any CNS-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the nervous system.
  • Nervous system-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a- 5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • MiRNA- binding sites from any pancreas-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the pancreas.
  • Pancreas-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2- 3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR- 562, MiRNA-binding sites from any kidney-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression
  • miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR- 143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • MiRNA-binding sites from any muscle-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the muscle.
  • Muscle-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
  • miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.
  • miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17- 3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-115p, miR-19b-2-5p, miR- 19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5
  • MiRNA-binding sites from any endothelial cell-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the endothelial cells.
  • miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR ⁇ 34c ⁇ 5p, miR ⁇ 449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let- 7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells.
  • MiRNA-binding mi
  • a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development, and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436; Goff LA et al., PLoS One, 2009, 4:e7192; Morin RD et al..
  • various cell lineages such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells
  • MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let.7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR- 1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR- 200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR
  • the binding sites of embryonic stem cell-specific miRNAs can be included in or removed from the 3'UTR of a polyribonucleotide of the disclosure to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a. degenerative condition (e.g, degenerative diseases), or to stimulate the senescence and. apoptosis of stem cells in a disease condition (e.g., cancer stem cells).
  • a degenerative condition e.g, degenerative diseases
  • apoptosis of stem cells in a disease condition e.g., cancer stem cells.
  • miRNAs are differentially expressed in cancer cells (W02008/154098, US2013/0059015, US2013/0042333, WO201 1/157294), cancer stern cells (US2012/0053224); pancreatic cancers and diseases (US2009/0131348, 1182011/0171646, US2010/0286232, US8389210); asthma, and inflammation (US8415096); prostate cancer (US2013/0053264); hepatocellular carcinoma (WO2012/151212, US2012/0329672, W02008/054828, US8252538); lung cancer cells (WO2011/076143, WO2013/033640, W02009/070653, US2010/0323357); cutaneous T cell lymphoma (W02013/011378); colore
  • miRNA-binding sites for miRNAs that are over-expressed in certain cancer and/or tumor cells can be removed from the 3'UTR of a polyribonucleotide of the disclosure, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death. Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected.
  • MiRNA can also regulate complex biological processes such as angiogenesis (e.g, miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176).
  • angiogenesis e.g, miR-132
  • miRNA-binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polyribonucleotides (or polyribonucleotides encoded by polynucleotides) to biologically relevant cell types or relevant biological processes.
  • the polyribonucleotides of the disclosure are defined as auxotrophic polyribonucleotides.
  • the therapeutic and/or prophylactic agent is a peptide therapeutic agent.
  • the therapeutic and/or prophylactic agent is a polypeptide therapeutic agent.
  • the therapeutic agent is a protein.
  • the therapeutic agent can be delivered to (administered to) a subject, organ, tissue or cell using the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure as an encoding polynucleotide that is translated (and optionally transcribed if DNA) into the therapeutic polypeptide after delivery.
  • the peptide or polypeptide is naturally derived, e.g, isolated from a natural source.
  • the peptide or polypeptide is a synthetic molecule, e.g. , a. synthetic peptide or polypeptide produced in vitro.
  • the peptide or polypeptide is a recombinant molecule.
  • the peptide or polypeptide is a chimeric molecule.
  • the peptide or polypeptide is a fusion molecule.
  • the peptide or polypeptide therapeutic agent of the composition is a naturally occurring peptide or polypeptide.
  • the peptide or polypeptide therapeutic agent of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g, contains less than 3, less than 5, less than 10, less than 15, less than 20, or less than 25 amino substitutions, deletions, or additions compared to its wild type, naturally occurring peptide or polypeptide counterpart).
  • the protein comprises a wild type, or substantially wiki type protein, or a protein which retains substantially wild type activity, that is administered to a subject with a disease characterized by a deficiency in the expression or activity of the protein.
  • the disease is a rare disease, i.e. a disease that effects a small proportion of the population.
  • rare diseases are caused by mutations in autosomal recessive genes that, lead to loss of gene activity , and can thus be treated by supplying the missing gene activity.
  • the lipid nanoparticles, pharmaceutical compositions and. methods of the disclosure can be used to supply the protein product from the mutated gene, or a DNA or mRNA encoding the protein.
  • the disease compri ses Propionic acidemia (PA), Methylmalonic acidemia (MMA), Glycogen Storage Disease Type la (GSDla), Ornithine transcarbamylase deficiency (OTC), Phenylketonuria (PKU), or Crigler-Najjar Syndrome Type 1 (CN-1).
  • PA Propionic acidemia
  • MMA Methylmalonic acidemia
  • GSDla Glycogen Storage Disease Type la
  • OTC Ornithine transcarbamylase deficiency
  • PKU Phenylketonuria
  • CN-1 Crigler-Najjar Syndrome Type 1
  • the disease comprises Propionic acidemia (PA), and the protein comprises rpropionyl-CoA carboxylase subunit alpha. (PCC A) and/or propionyl-CoA carboxylase subunit beta (PCCB), or a variant or derivative thereof.
  • PA Propionic acidemia
  • PCC A rpropionyl-CoA carboxylase subunit alpha.
  • PCCB propionyl-CoA carboxylase subunit beta
  • the disease comprises Methylmalonic acidemia (MMA), and the protein comprises methylmalonyl-CoA mutase (MMUT).
  • MMA Methylmalonic acidemia
  • MMUT methylmalonyl-CoA mutase
  • the disease comprises Glycogen Storage Disease Type la (GSDla), and the protein comprises enzyme glucose-6-phosphatase (G6Pase), or a. derivative or variant thereof.
  • GSDla Glycogen Storage Disease Type la
  • G6Pase enzyme glucose-6-phosphatase
  • the disease comprises Ornithine transcarbamylase deficiency
  • the protein comprises ornithine transcarbamylase (OTC), or a variant or derivative thereof.
  • OTC ornithine transcarbamylase
  • the disease comprises Phenylketonuria. (PKU)
  • the protein comprises phenylalanine hydroxylase (PAH), or a variant or derivative thereof.
  • the disease comprises Crigler-Najjar Syndrome Type 1 (CN-I), and the protein comprises UDP glucuronosyltransferase family 1 member Al (UGT1A1), or a variant or derivative thereof.
  • CN-I Crigler-Najjar Syndrome Type 1
  • UDT1A1 UDP glucuronosyltransferase family 1 member Al
  • the polypeptide comprises a fusion protein.
  • fusion proteins include, but are not limited, to, fusion proteins comprising DNA binding domains (e.g., TALEN, or transcription activator-like effector nuclease DNA. binding domains. Zinc Finger Nuclease, or ZFP DNA binding domains, or catalytically inactive Class 2 CRISPR/Cas systems) fused to one or more effector domains, such as transcriptional activation, repression or base editing domains, and the like.
  • DNA binding domains e.g., TALEN, or transcription activator-like effector nuclease DNA. binding domains. Zinc Finger Nuclease, or ZFP DNA binding domains, or catalytically inactive Class 2 CRISPR/Cas systems
  • fusion proteins include proteins with fusion domains to enhance in vivo stability and/or bioavailable of the protein (e.g., Fc fusion proteins), and fusion proteins with enhanced tissue, organ or cellular specificity (e.g., antibody fusion proteins).
  • the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure can be used to deliver (administer) gene editing system to a subject organ, tissue or cell, thereby editing the genome of the target cell, or cells of the subject, organ or tissue.
  • Suitable gene editing systems will be known to persons of ordinary skill in the art, and include, but are not limited to, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems.
  • ZFNs Zinc Finger Nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR/Cas systems CRISPR/Cas systems.
  • CRISPR/Cas system refers to genome editing systems derived from adaptive immune systems present in bacteria and archaea.
  • a typical CRISPR/Cas system has two components: an effector protein (or protein complex, depending on the system) which can cleave a target nucleic acid sequence, and a. guide nucleic acid (or nucleic acids), usually RNA, which contain a targeting sequence complementary' to the target sequence and which bind the effector protein(s) and guide the effector protein(s) to the corresponding target.
  • the guide nucleic acid can be one nucleic acid (a single gNA) or multiple nucleic acids (e.g., a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA)) depending on the CRISPR/Cas system.
  • a gNA typically includes a scaffold sequence, which interacts with the CRISPR protein, and an approximately 20 nucleotide targeting sequence that is complementary to, and capable of hybridizing to, the target sequence.
  • CRISPR/Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and several subtypes, with multi-Cas protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI) (see, for example, Li et al. Comput Struct Biotechnol J. 2020 Sep 8,18:2401 -2415, the contents of which are incorporated by reference herein in their entirety).
  • the nuclease activity of the CRISPR/Cas system can be modified or eliminated to produce a CRISPR/Cas system capable of binding, but not cleaving, or nicking rather than cleaving, a target nucleic acid.
  • Such catalytically inactive CRISPR/Cas systems have utility, for example as DNA binding domains incorporated into fusion proteins.
  • CRISPR/Cas systems derived from bacteriophages (e.g., CasPhi) are also contemplated as within the scope of the instant disclosure.
  • Selection of suitable guide nucleic acid sequences (e.g., gRNA) and targeting sequences based on the proto-spacer adjacent motif (PAM) specificity of the Cas protein are within the skill of persons of ordinary skill in the art.
  • the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure deliver (administer) a nucleic acid to a subject, organ, tissue or cell that, is part of a gene editing system, such as the CRISPR/Cas systems described herein.
  • the nucleic acid comprises at least one nucleic acid suitable for a genome editing technique selected from the group consisting of an mRNA. encoding a gene editing protein (e.g. a ZFN, TALEN or CRISPR protein), a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template, or a combination thereof.
  • a gene editing protein e.g. a ZFN, TALEN or CRISPR protein
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • sgRNA single guide RNA
  • DNA repair template or a combination thereof.
  • the nucleic acid comprises an mRNA encoding a protein of a gene editing system, such as a CRISPR protein.
  • the nucleic acid comprises DNA encoding the gene editing protein, such as a. CRISPR protein.
  • the nucleic acid comprises a gNA (or a component of gNA, where the gNA is not a. single gNA).
  • the gene editing comprises use of a DNA repair pathway.
  • DNA repair pathways which occur after double-strand breaks in the target DNA molecule, include, but are not limited to, homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA).
  • the DNA repair is comprises a template (e.g., HR).
  • the gene editing system comprises a DNA template molecule (e.g., one which includes a wild type version or desirable mutation, such as an insertion, deletion or base-pair substitution), of the target DNA to be edited.
  • the DNA template molecule is delivered to (administered to) the subject, organ, tissue or cell using the lipid nanoparticles, pharmaceutical compositions and. methods of the disclosure.
  • the gene editing system comprises a modified gene editing system that is catalytically inactive, and can be used for epigenetic regulation, rather than editing of, a target nucleic acid in a. subject, organ, tissue or cell.
  • the CRISPR protein can be catalytically inactivated and fused to an effector domain, or the DNA binding domain of the ZFN or TALEN can be fused to a domain other than a nuclease domain.
  • the DNA binding domains of the gene editing systems described herein can be fused to one or more transcriptional activator domains (e.g., VP16) or repressor domains (KRAB, SETDB1, DNMT1, DMT3A and the like).
  • multiple components of the gene editing system e.g., the CRISPR protein, or an mRNA or DNA encoding same, as well as the gNA, and optionally the DNA template molecule
  • the components of the gene editing system can be incorporated into different sialic lipid nanoparticles.
  • the disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle or pharmaceutical composition of the disclosure.
  • mRNA messenger ribonucleic acid
  • the lipid nanoparticle comprises Compound 9.
  • the mRNA can comprise an open reading frame encoding an antigen, for example a cancer antigen or an infectious disease antigen as described herein.
  • an antigen for example a cancer antigen or an infectious disease antigen as described herein.
  • the antigen comprises an infectious disease antigen, for example a virus or bacteria.
  • the infectious disease antigen comprises a protein, or a. portion of a protein, from the agent (bacteria, virus, parasitic eukaryote etc.) to which an immune response is desired.
  • the infectious disease comprises an infectious disease that primarily affects adults, an infectious disease that primarily affects children and/or adolescents (adolescent and pediatric infectious disease), or an infectious disease that affects both adults and children.
  • the infectious diseases comprises a coronavirus (e.g., SARS-CoV-2 virus, which causes COVID-19), an influenza, virus (e.g., influenza. A, B, C or D), a respiratory syncytial virus (RSV), or a human metapneumovirus (hMPV).
  • the infectious disease comprises disease caused by a latent virus, i.e. a virus that is present in a resting state in the body.
  • the latent virus comprises a cytomegalovirus (CMV), an Epstein-Barr virus (EBV), a herpes simplex vims (HSV), e.g. HSV type 2, a varicella zoster vims (VZV), or a. human immunodeficiency vims (HIV) such as HIV-1 or HIV-2.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • HSV herpes simplex vims
  • VZV varicella zoster vims
  • HIV human immunodeficiency vims
  • the infectious disease comprises an enteric virus, for example a norovirus.
  • the infectious disease comprises a bacteria.
  • Exemplary bacteria include, but are not limited to Borrelia burgdorferi, which cause lyme disease.
  • the disease is a disease that has caused, or has the potential to cause, a public health crisis due to the ease with which such viruses spread in the population and the severity of the illness such viruses cause.
  • viruses such as Zika virus, Nipah virus and monkeypox (Mpox) have all caused, or have the potential to cause public health problems.
  • the virus is a strain of Influenza A or Influenza B or combinations thereof.
  • the antigenic polypeptide encodes a hemagglutinin protein or fragment thereof.
  • the hemagglutinin protein is , or a portion thereof.
  • the virus comprises coronavirus (e.g., SARS-CoV-2 virus and variants or strains thereof), and the antigen comprises the coronavirus spike protein (S) or a portion thereof.
  • SARS-CoV-2 antigens are described, for example, in WO2022155524, the contents of which are incorporated by reference in their entirety.
  • the virus comprises RSV or hMPV
  • the antigen comprises a viral polyprotein or a portion thereof. Additional RSV and hMPV antigens are described in US20180326045, the contents of which are incorporated by reference in their entirety.
  • the virus comprises CMV
  • the antigen comprises one or more CMV glycoproteins.
  • the antigen comprises cancer antigen.
  • cancer antigens to which immune responses can be directed are known in the art.
  • tumor antigens are molecules found in tumor cells that trigger an immune response in the subject with the tumor.
  • Tumor antigens can be tumor associated antigens (TAAs), which are expressed in healthy tissues but overexpressed in cancer cells, or tumor specific antigens (TSA), which are only found in cancer cells.
  • TAAs tumor associated antigens
  • TSA tumor specific antigens
  • the cancer antigen (and resulting vaccine) is individualized, i.e. derived from and specific to the cancer of the subject.
  • Individualized vaccines may include RNA encoding for one or more known cancer antigens specific for the tumor or cancer antigens specific for each subject, referred to as neoepitopes or patient specific epitopes or antigens.
  • a "patient specific cancer antigen” is an antigen that has been identified as being expressed in a. tumor of a particular patient. The patient specific cancer antigen may or may not be typically present in tumor samples generally.
  • Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced in comparison to that in cancerous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes.
  • Exemplary cancers that can be treated with individualized cancer vaccines include, but are not limited to, melanoma, nonsmall cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), renal cell carcinoma (R.CC), bladder cancer and solid tumors (early and late).
  • the cancer antigen comprises a checkpoint antigen.
  • Vaccines to checkpoint antigens can stimulate effector T cells that target and kill suppressive immune and cancer cells that express high levels of checkpoint.
  • the checkpoint antigen comprises IDO (indoleamine 2,3-dioxygenase 1) or a fragment thereof, PD-L1 (CD274 molecule), or a. fragment thereof, or combinations thereof.
  • the antigen or epitope is based on specific mutations (neoepitopes) and those expressed by cancer- germline genes (antigens common to tumors found in multiple patients).
  • An epitope also known as an antigenic determinant, as used herein, is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells.
  • Epitopes include B cell epitopes and T cell epitopes.
  • B-cell epitopes are peptide sequences which are required for recognition by specific antibody producing B -cells.
  • B cell epitopes refer to a specific region of the antigen that is recognized by an antibody.
  • the portion of an antibody that binds to the epitope is called a paratope.
  • An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope.
  • a linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein.
  • the sequences that interact with the antibody- are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide.
  • Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous, i.e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.
  • T-cell epitopes are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I.
  • the peptide epitope may be any length that is reasonable for an epitope.
  • the peptide epitope is 9-30 amino acids. In other
  • the length is 9- 22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21, 10-20, 1 1 -22, 22-21, 1 1 -20, 12-22, 12-21, 12-20,13-22, 13-21, 13- 20, 14-19, 15-18, or 16-17 amino acids.
  • the of the disclosure may include mRNA sequences encoding one or more antigens.
  • the mRNA comprises the sequence of 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more antigens.
  • the mRNA comprises sequences of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less cancer antigens.
  • the mRNA comprises sequences of 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40- 100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15- 50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100- 500, 50-500, 50-800, 50-1,000, or 100-1,000 antigens.
  • a lipid nanoparticle may include one or more components in addition to those described in the preceding sections.
  • a lipid nanoparticle may include one or more small hydrophobic molecules such as a vitamin (e.g, vitamin A or vitamin E) or a. sterol .
  • Lipid nanoparticles may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components.
  • a permeability enhancer molecule may be a molecule described by U.S. patent application publication No. 2005/0222064, for example.
  • Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g, glycogen and derivatives and analogs thereof).
  • a polymer may be included in and/or used to encapsulate or partially encapsulate a lipid nanoparticle.
  • a polymer may be biodegradable and/or biocompatible.
  • a polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLG.A), poly(L -lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L- lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-la.ctide), poly(D,L- lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PEL), hydroxypropyl
  • PCL
  • Surface altering agents may include, but are not limited to, anionic proteins (e.g. , bovine serum albumin), surfactants (e.g, cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g, cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin ⁇ 4, dornase alfa, neltenexine, and erdosteine), and DNases (
  • a lipid nanoparticle may also comprise one or more functionalized lipids.
  • a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction.
  • a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging.
  • the surface of a lipid, nanoparticle may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.
  • lipid nanoparticles may include any substance useful in pharmaceutical compositions.
  • the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art. (See, for example, Remington’s The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.)
  • diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and/or combinations thereof.
  • Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • crospovidone cross-
  • Surface active agents and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g, bentonite [aluminum silicate] and.
  • natural emulsifiers e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g, bentonite [aluminum silicate] and.
  • VEEGUM® [magnesium aluminum silicate]
  • long chain amino acid derivatives long chain amino acid derivatives
  • high molecular weight alcohols e.g, stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, propylene glycol monostearate, and polyvinyl alcohol
  • carbomers e.g, carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer
  • carrageenan cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and methylcellulose), sorbitan fatty acid, esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate
  • a binding agent may be starch (e.g., cornstarch and starch paste), gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, and mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti.
  • starch e.g., cornstarch and starch paste
  • sugars e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, and mannitol
  • natural and synthetic gums e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti.
  • gum mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol, inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.
  • VEEGUM® magnesium aluminum silicate
  • preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chiorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SEES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II NEOLONETM, KATHONTM, and/or EUXYL®.
  • buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., H
  • Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana,
  • the present disclosure provides a. method of preparing the population of lipid nanoparticles described herein.
  • the method comprises: i) mixing an ionizable lipid, a structural lipid, and a phospholipid, with a first buffer, thereby forming a population of intermediate empty lipid nanoparticles.
  • the method comprises: i) mixing an ionizable lipid, a structural lipid, a phospholipid, and a PEG lipid, with a first buffer, thereby forming a population of intermediate empty lipid nanoparticles.
  • the method further comprises: ii) adding a second buffer to the intermediate empty lipid nanoparticles, thereby forming a population of empty lipid nanoparticles.
  • the method further comprises: iii) mixing a therapeutic agent (e.g., a nucleic acid) with the empty-lipid nanoparticles, thereby forming a population of loaded lipid nanoparticles.
  • a therapeutic agent e.g., a nucleic acid
  • the method further comprises processing the empty lipid nanoparticles or the loaded lipid nanoparticles.
  • the step of processing comprises: a) adding a cryoprotectant to the empty lipid nanoparticles or the loaded lipid nanopartides; b) lyophilizing the empty lipid nanoparticles or the loaded lipid nanoparticles; c) storing the lyophilized empty lipid nanoparticles or the lyophilized loaded lipid nanoparticles; and/or d) adding a buffering solution to the lyophilized empty lipid nanoparticles or the lyophilized loaded lipid nanoparticles.
  • the present disclosure provides a. pharmaceutical composition, comprising the population of lipid nanoparticles described herein, and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical composition is free of therapeutic agent (e.g, RNA).
  • the pharmaceutical composition comprises a. therapeutic agent (e.g., RNA).
  • a. therapeutic agent e.g., RNA
  • compositions may include one or more lipid nanoparticles.
  • a pharmaceutical composition may include one or more lipid nanoparticles including one or more different therapeutics and/or prophylactics.
  • Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein.
  • General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21 st Edition, A.. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.
  • Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a.
  • lipid nanoparticle in the formulation of the disclosure may be incompatible with a component of a. lipid nanoparticle of the formulation if its combination with the component or lipid nanoparticle may result in any undesirable biological effect or otherwise deleterious effect.
  • one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid nanoparticle.
  • the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention.
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved, for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United. States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • lipid nanoparticles Relative amounts of the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • a pharmaceutical composition comprises between 0.1% and 100% (wt/wt) of one or more lipid nanoparticles.
  • a pharmaceutical composition comprises between 0.1% and 15% (wt/vol) of one or more amphiphilic polymers (e.g, 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w/v).
  • the lipid nanoparticles and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g, being stored at a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30
  • the pharmaceutical composition comprising one or more lipid, nanoparticles is a solution or solid (e.g, via lyophilization) that, is refrigerated for storage and/or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C.
  • the disclosure also relates to a method of increasing stability of the lipid nanoparticles and by storing the lipid nanoparticles and/or pharmaceutical compositions thereof at. a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C
  • Lipid nanoparticles and/or pharmaceutical compositions including one or more lipid nanoparticles may be administered, to any patient or subject including those patients or subjects that may benefit from a. therapeutic effect provided by the delivery of a therapeutic and/or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system.
  • lipid nanoparticles and pharmaceutical compositions including lipid nanoparticles are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal.
  • compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary , if any, experimentation.
  • Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and/or rats.
  • a pharmaceutical composition including one or more lipid nanoparticles may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g, lipid nanoparticle).
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.
  • compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration.
  • pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and. granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.
  • liquid dosage forms e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs
  • injectable forms e.g., solid dosage forms (e.g., capsules, tablets, pills, powders, and. granules)
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms comprise inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol,
  • oral compositions can include additional therapeutics and/or prophylactics, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3 -butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed, as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • biodegradabl e polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients, such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and. therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and. therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules.
  • an active ingredient is mixed with at least- one inert, pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g, cetyl alcohol and glycerol), e.g.
  • lubricants e.g, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate
  • the dosage form may comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a. composition that they release the active ingredient(s) only. In some embodiments, the solid compositions may optionally comprise opacifying agents and. can be of a.
  • compositions that, they release the active ingredient(s) in a certain part, of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches.
  • an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
  • the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled, delivery of a compound, to the body.
  • dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
  • rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662.
  • Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof.
  • Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis are suitable.
  • Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144, 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941 ,880, 4,940,460; and PCT publications WO 97/37705 and WO 97/13537.
  • Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. .
  • conventional syringes may be used in the classical mantoux method of intradermal administration.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations, such as liniments, lotions, oil in water and/or water in oil emulsions, such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
  • Topically administrable formulations may, for example, comprise from about 1% to about 10% (wt/wt) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self- propelling solvent/powder dispensing container, such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Dry' powder compositions may include a solid fine powder diluent, such as sugar, and. are conveniently provided in a. unit dose form.
  • Low-boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50% to 99.9% (wt/wt) of the composition, and active ingredient may constitute 0.1% to 20% (wt/wt) of the composition.
  • a propellant may further comprise additional ingredients, such as a liquid non- ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
  • Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a. flavoring agent, such as saccharin sodium, a volatile oil, a buffering agent, a surface-active agent, and/or a preservative, such as methylhydroxybenzoate.
  • Droplets provided by this route of administration may have an average diameter in the range from about 1 nm to about 200 nm.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ m to 500 ⁇ m. Such a formulation is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (wt/wt) and as much as 100% (wt/wt) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may- be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may comprise, for example, 0.1% to 20% (wt/wt) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0. 1/1.0% (wt/wt) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
  • Other ophthalmically administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this present disclosure.
  • the present disclosure provides methods of producing a polypeptide of interest in a. mammalian cell.
  • Methods of producing polypeptides involve contacting a cell with a formulation of the disclosure comprising a. lipid nanoparticle including an mRNA encoding the polypeptide of interest.
  • the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.
  • the polypeptide of interest is a protein.
  • the step of contacting a mammalian cell with a. lipid nanoparticle including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro.
  • the amount of lipid nanoparticle contacted with a cell, and/or the amount of mRNA. therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the lipid nanoparticle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors.
  • an effective amount of the lipid nanoparticle will allow for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.
  • the step of contacting a lipid nanoparticle including an mRNA. with a cell may involve or cause transfection.
  • a phospholipid including in the lipid component of a lipid nanoparticle may facilitate transfection and/or increase transfection efficiency, for example, by interacting and/or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell.
  • the lipid nanoparticles described herein may be used therapeutically or prophylactically.
  • an mRNA included in a lipid, nanoparticle may encode a therapeutic polypeptide (e.g, in a translatable region) and produce the therapeutic polypeptide upon contacting and/or entry (e.g., transfection) into a cell.
  • an mRNA included in a lipid nanoparticle may encode a polypeptide that may improve or increase the immunity of a. subject.
  • contacting a cell with a lipid nanoparticle comprising a sialic acid lipid may effectuate a reduced inflammatory response, as measured by expression of Sca- 1, which is a surrogate marker of inflammatory reaction, as compared to the inflammatory' response effectuated by a lipid nanoparticle that does not comprise a sialic acid lipid.
  • the present disclosure provides a method of delivering a therapeutic agent to a cell in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to a cell in a subject.
  • the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to a cell in a subject.
  • the LNPs of the present disclosure can be used to deliver nucleic acid payloads to cell populations in which expression or function of such nucleic acid molecules is desired.
  • the subject LNPs represent an improvement over alternative delivery vehicles (e.g., LNPs of alternative composition) as they deliver payloads to desired cell lines and yet do not increase expression of markers known to be associated with inflammation, e.g., Sca-1.
  • the LNPs of the present disclosure are used to deliver nucleic acid payload molecules to immune cells, e.g., to myeloid and lymphoid cells.
  • payload molecules are delivered to hematopoietic stem and progenitor cell (HSPC) populations.
  • HSPC hematopoietic stem and progenitor cell
  • the LNPs of the present disclosure can be used to deliver payload to the group(s) of cells which are referred to variously in the art and include: multipotent progenitors ; hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-; hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS); LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin- cKit+Scal-)); and.
  • HSCs hematopoietic stem cells
  • HSPCs hematopoietic stem progenitor cells
  • LKS hematopoietic stem progenitor cells
  • LK cells i.e., lineage (negative)
  • EPCs Erythroid progenitor cells
  • the subject LNPs are used to deliver mRNA payload to mature myeloid cells, e.g., monocytes and macrophages. In another embodiment, the subject LNPs are used to deliver mRNA payload to macrophages.
  • LNPs of the present disclosure may comprising a sialic acid lipid, an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid.
  • LNPs comprising a sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to a multipotent progenitor cell.
  • LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin- cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin- cKit+Scal-)
  • LNPs comprising a sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • the LNPs comprise a combination of ionizable lipid, PEG-lipid, sialic acid lipid, and phospholipid as specified in Table 1A”:
  • Exemplary embodiments of LNPs of the present disclosure include those specified in
  • type A’ LNPs are used to deliver art mRNA payload to a multipotent progenitor cell.
  • type A’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type A’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type A’ LNPs are used to deliver an mRNA payload to LK cells
  • type A.’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type B’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type B’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type B’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and. multipotent progenitors, (also known as LKS).
  • type B’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type B’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type E’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type E’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type E’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type E’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type E’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type F’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type F’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type F’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type F’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type F’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type G’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type G’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • type G’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type G’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type G’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type J’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type J’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type J’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type J’ LNPs are used to deliver an mRN A payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type J’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type K’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type K’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type K’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type K’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type K’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
  • EPCs Erythroid progenitor cells
  • type L’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type L’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type L’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type L’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type L’ LNPs are used to deliver an mRNA payload to Erythroid, progenitor cells (EPCs).
  • EPCs Erythroid, progenitor cells
  • type M’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type M’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type M’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multipotent progenitors
  • type M’ LNPs are used to deliver an mRNA payload to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • LK cells i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
  • type M’ LNPs are used to deliver an mRNA payload to Erythroid, progenitor cells (EPCs).
  • EPCs Erythroid, progenitor cells
  • type N’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
  • type N' LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
  • HSCs hematopoietic stem cells
  • LT-HSCs long term HSCs
  • type N’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multi potent progenitors, (also known as LKS).
  • HSPCs hematopoietic stem progenitor cells
  • LKS multi potent progenitors
  • type N’ LNPs are used to deliver an mRNA payload, to LK cells (i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal -)).
  • type N’ L'NPs are used to deliver an mRNA payload to Eiythroid progenitor cells (EPCs).
  • the present disclosure provides a method of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject, the population of lipid nanoparticles or pharmaceutical composition described herein.
  • HSPC hematopoietic stem and progenitor cell
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to hematopoietic stem and progenitor cells (HSPC) in a subject.
  • HSPC hematopoietic stem and progenitor cells
  • the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to hematopoietic stem and progenitor cells (HSPC) in a subject.
  • the present disclosure provides a method of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a. subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
  • EPC erythroid progenitor cell
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to an eiythroid progenitor cells (EPC) in a subject.
  • EPC eiythroid progenitor cells
  • the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to eiythroid progenitor cells (EPC) in a subject.
  • EPC eiythroid progenitor cells
  • the present disclosure provides a method of delivering a therapeutic agent to myeloid cells in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to myeloid cells in a subject.
  • the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to myeloid cells in a subject.
  • the present disclosure provides a method of delivering a therapeutic agent to lymphoid cells in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to lymphoid cells in a subject.
  • the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to lymphoid cells in a subject.
  • the subject is human.
  • the present disclosure provides methods of delivering a therapeutic and/or prophylactic, such as a nucleic acid, to a mammalian cell or organ. Delivery- of a therapeutic and/or prophylactic to a cell involves administering a formulation of the disclosure that comprises a lipid nanoparticle including the therapeutic and/or prophylactic, such as a nucleic acid, to a subject, where administration of the composition involves contacting the cell with the composition.
  • nucleic acid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ.
  • a translatable mRNA upon contacting a cell with the lipid nanoparticle, may be translated in the cell to produce a polypeptide of interest, e.g., a protein.
  • a lipid nanoparticle may target a particular type or class of cells (e.g, cells of a particular organ or system thereof).
  • a lipid nanoparticle including a therapeutic and/or prophylactic of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, bone marrow, or lung.
  • Specific delivery to a particular class of cells, an organ, or a. system or group thereof implies that a. higher proportion of lipid nanoparticles including a therapeutic and/or prophylactic are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of a lipid nanoparticle to a mammal.
  • tissue of the targeted destination e.g., tissue of interest, such as a liver
  • another destination e.g, the spleen
  • the tissue of interest is selected from the group consisting of a liver, kidney, bone marrow, a lung, a spleen, a femur, vascular endothelium in vessels (e.g, intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g, via intratumoral injection).
  • the present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need. thereof the population of lipid nanoparticles or pharmaceutical composition described herein (e.g., in a therapeutically effective amount).
  • the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described, herein for use in treating or preventing a disease or disorder in a subject.
  • the present disclosure provides use of the population of lipid nanoparticles or pharmaceutical composition described herein in the manufacture of a medicament for treating or preventing a disease or disorder.
  • the population of lipid nanoparticles or pharmaceutical composition is administered parenterally.

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Abstract

The present disclosure provides lipids comprising a sialic acid moiety (sialic acid lipids) and lipid nanoparticles suitable for delivery of therapeutic agents to hematopoietic stem and progenitor cells (HSPCs) and myleloid and lymphoid cells, wherein the lipid nanoparticles comprise a sialic acid lipid. The present disclosure also provides therapeutic and diagnostic uses related to the lipid nanoparticles. The sialic acid lipids have formula (SA-1).

Description

LIPID NANOPARTICLE COMPOSITIONS COMPRISING SIALIC ACID
DERIVATIVES AND THE USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/627,653, filed on January 31, 2024, the contents of which are incorporated by reference herein in their entirety for all purposes.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0002] The contents of the electronic sequence listing (MRNA-166_001WO__SeqList_ST26,xml; Size: 9,556 bytes; and Date of Creation: January 29, 2025) are herein incorporated by reference in its entirety.
BACKGROUND
[0003] The effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge. In particular, the delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of such species. Thus, there exists a need to develop methods and compositions to facilitate the delivery of therapeutics and prophylactics such as nucleic acids to cells. In addition, delivery vehicles that can both promote desirable immune responses while dampening undesirable immune responses would be of great benefit.
[0004] Lapid nanoparticles have proven effective as transport vehicles into cells and/or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids. Though a variety of such lipid-containing nanoparticles have been demonstrated, improvements in safety, efficacy, and specificity are still needed.
SUMMARY
[0005] In some aspects, the present disclosure provides a sialic acid lipid of Formula (SA-I): or a salt or ionized form thereof.
[0006] In some aspects, the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid described herein.
[0007] In some aspects, the present disclosure provides a population of lipid nanoparticles comprising a sialic acid lipid, an ionizable lipid, and a structural lipid, wherein the sialic acid lipid of Formula (SA-1):
(SA-1) or a salt or ionized form thereof.
[0008] In some aspects, the present disclosure provides methods of preparing the lipid nanoparticle and the population of lipid nanoparticles described herein.
[0009] In some aspects, the present disclosure provides pharmaceutical compositions comprising the lipid nanoparticle, pharmaceutical compositions comprising the population of lipid nanoparticles described herein, and methods of preparing the pharmaceutical compositions.
[0010] In some aspects, the disclosure provides methods of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
[0011] In some aspects, the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in delivering therapeutic agents to hematopoietic stem and progenitor cells (HSPC) in subjects.
[0012] In some aspects, the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of medicaments for delivering therapeutic agents to hematopoietic stem and progenitor cells (HSPC) in subjects. [0013] In some aspects, the disclosure provides methods of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
[0014] In some aspects, the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in delivering therapeutic agents to erythroid progenitor cells (EPC) in subjects.
[0015] In some aspects, the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of medicaments for delivering therapeutic agents to erythroid progenitor cell (EPC) in subjects.
[0016] In some aspects, the disclosure provides methods of delivering a therapeutic agent to a myeloid cell in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
[0017] In some aspects, the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition for use in delivering therapeutic agents to myeloid cells in subjects,
[0018] In some aspects, the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition in the manufacture of medicaments for delivering therapeutic agents to myeloid cells in subjects.
[0019] In some aspects, the disclosure provides methods of delivering a therapeutic agent to a lymophoid cell in a subject, comprising administering to the subject a lipid nanoparticle or a population of lipid nanoparticles described herein.
[0020] In some aspects, the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition for use in delivering therapeutic agents to lymophoid cells in subjects.
[0021] In some aspects, the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical composition in the manufacture of medicaments for delivering therapeutic agents to lymophoid cells in subjects.
[0022] In some aspects, the present disclosure provides lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions for use in treating or preventing diseases or disorders in subjects. [0023] In some aspects, the disclosure provides uses of lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions in the manufacture of a medicaments for treating or preventing diseases and disorders in subjects.
[0024] In some aspects, the present disclosure provides nanoparticles prepared by the processes of preparing nanoparticles provided herein.
[0025] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein.
[0026] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein the mRNA comprises an open reading frame encoding a cancer antigen or an infectious disease antigen.
[0027] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (li) the vaccine comprises a mRNA comprising an open reading frame encoding a cancer antigen or an infectious disease antigen.
[0028] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises (i-i) a sialic acid disclosed herein (e.g., Compound No. 9), (i-ii) an ionizable lipid disclosed herein, (i-iii) a structural lipid disclosed herein, (i-iv) a phospholipid disclosed herein, and (i-v) a PEG-lipid disclosed herein, and (ii) the mRNA comprises an open reading frame encoding a cancer antigen or an infectious disease antigen.
[0029] In some aspects, the present disclosure provides a method of reducing susceptibility to or symptoms of an infectious disease in a subject, comprising administering a vaccine disclosed herein to the subject, wherein the vaccine comprises an mRNA comprising an open reading frame encoding an infectious disease antigen.
[0030] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in a method of reducing susceptibility to or symptoms of an infectious disease in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRN A comprises an open reading frame encoding an infectious disease antigen. [0031] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in the preparation of a medicament for reducing susceptibility to or symptoms of an infectious disease in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding an infectious disease antigen.
[0032] In some aspects, the present disclosure provides the use of a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding an infectious disease antigen, for reducing susceptibility to or symptoms of an infectious disease in a subject.
[0033] In some aspects, the present disclosure provides a method treating cancer in a subject, comprising administering a vaccine disclosed herein to the subject, wherein the mRNA comprises an open reading frame encoding a cancer antigen.
[0034] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in a method of treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
[0035] In some aspects, the present disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for use in the preparation of a medicament for treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
[0036] In some aspects, the present, disclosure provides the use of a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle disclosed herein for treating cancer in a subject, wherein (i) the lipid nanoparticle comprises a sialic acid disclosed herein (e.g., Compound No. 9), and (ii) the mRNA comprises an open reading frame encoding a cancer antigen.
[0037] In some aspects, the present disclosure provides a composition comprising (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle disclosed herein.
[0038] In some aspects, the present disclosure provides a composition comprising: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle comprising (iii-i) an ionizable lipid disclosed herein, (iii-ii) a PEG lipid disclosed herein, (iii-iii) a structural lipid disclosed herein, (iii-iv) a phospholipid disclosed herein, and (iii- v) a sialic acid lipid disclosed herein (e.g., Compound No. 1).
[0039] In some aspects, the present disclosure provides a method of editing a sequence of a target nucleic acid in a cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle disclosed herein.
[0040] In some aspects, the present disclosure provides a method of editing a sequence of a target nucleic acid in a cell, preferably a human cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding a CRISPR nuclease, and/or (ii) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and (iii) a lipid nanoparticle comprising (iii-i) an ionizable lipid disclosed herein, (iii-ii) a PEG lipid disclosed herein, (iii-iii) a structural lipid disclosed herein, (iii-iv) a phospholipid disclosed herein, and (iii-v) a sialic acid lipid disclosed herein (e.g., Compound No. 1).
[0041] In some aspects, the present disclosure provides a method of delivering a polypeptide to a cell, preferably a human cell, comprising contacting the cell with a composition, wherein the composition comprises: (i) a nucleic acid comprising a sequence encoding the polypeptide; and (ii) a lipid nanoparticle comprising (ii-i) an ionizable lipid disclosed herein, (ii-ii) a PEG lipid disclosed herein, (ii-iii) a structural lipid disclosed herein, (ii-iv) a phospholipid disclosed herein, and (iii-v) a sialic acid lipid disclosed herein. In some aspects, the polypeptide comprises a therapeutic agent.
[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Ail publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
[0043] Other features and advantages of the disclosure will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 shows a flow-chart of PIPA process where sialic acid lipids were incorporated in the lipid stock solution.
[0045] FIG. 2 shows a flow-chart of PIPA process where sialic acid lipids were added in the PI stage.
[0046] FIG. 3 shows a HPLC chromatogram showing a distinct peak corresponding to compound 1 in an LNP produced by the process shown in FIG. 2.
[0047] FIG. 4 shows Octet Biolayer Interferometry (BLI) characterization of sialic acid LNPs. Human CD-33 is immobilized on NT A probes. All sialic acid containing LNPs showed significant binding to CD-33 compared to the control (bottom-most line).
[0048] FIG. 5 is a plot showing the percent of CD33 insertions/deletions (indels) in mouse bone marrow hematopoietic stem and progenitor cells after intravenous administration of sialic acid LNP containing spCas9 mRNA and an sgRNA targeting mouse CD33 to C57/B16 mice, with or without hematopoietic stem cell mobilization (M). Cells were lineage negative (lin-), and ckit receptor tyrosine kinase positive (ckit+).
[0049] FIGS. 6A-6C show the percentage of cells expressing green lantern after intravenous delivery of green lantern mRNA using the indicated sialic acid LNP to humanized female mice (Hu-CD34+NSG-SGM3). FIG. 6A shows expression of green lantern in my eloid progenitors cells (hCD45+, Lineage negative, CD34+ and CD38+). FIG. 6B shows green lantern expression in hematopoietic stem and progenitor cells (HSPCs) ( HCD45+, Lineage negative, CD34+ and CD38- ). FIG. 6B shows expression of green lantern in long-term repopulating hematopoietic stem cells (LT-HSCs) (hCD45+, Lineage negative, CD34+, CD38-, and CD45RA-). [0050] FIG. 7 is a diagram showing sialic acid LNP administration and sample collection for the experiments described in Example 8.
[0051] FIG. 8 is a plot showing average cell viability for cells collected from groups of mice administered sialic acid LNP as described in Example 8.
[0052] FIG. 9 is a plot showing average cell counts for cells collected from the spleens of the groups of mice administered sialic acid LNP as described in Example 8.
[0053] FIG. 10 is a plot showing the percentage of CD4+ T cells expressing, from left to right for each set of bars, Thl cytokines (IFNy, TNFa and IL-2), and Th2 cytokines (IL-4, IL-5 and IL- 15) for mice administered sialic acid containing LNPs as described in Example 8.
[0054] FIG. 11 is a plot showing the percentage of CD8+ T cells expressing, from left to right for each set of bars, CD 107α, IFNγ, TNFα and IL-2 for mice administered sialic acid containing LNPs as described in Example 8.
[0055] FIG. 12 is a plot showing influenza-specific antibody titers against influenza virus A/Sydney/5/2021 using sera from mice administered sialic acid containing LNPs as described in Example 8.
[0056] FIG. 13 is a plot showing total H1 Sydney IgG Titers at days 21 and 36 as described in Example 8.
[0057] FIG. 14 is a series of plots showing post-boost cytokine responses (Luminex, 6 hours post boost) for mice administered sialic acid containing LNPs as described in Example 8. Geometric mean responses and geometric 95% CI are shown for each group.
[0058] FIG. 15 is a series of plots showing post-boost, cytokine responses (Luminex, 6 hours post boost) for mice administered sialic acid containing LNPs as described in Example 8. Geometric mean responses and geometric 95% CI are shown for each group.
DETAILED DESCRIPTION
Compounds of the Present Disclosure
[0059] In an aspect, the present disclosure provides a sialic acid lipid of Formula (SA-I): or a salt or ionized form thereof, wherein:
M is *-O-C(=O)- or *-C(=O)-O-; wherein * indicates attachment to R;
R is C13-20 alkyl or C13-20 alkenyl;
M’ is *-O-C(=O)- or *-C(=O)-O-; wherein * indicates attachment to R’;
R’ is C13-20 alkyl or C13-20 alkenyl;
X is a pharmaceutically acceptable cation; n is 40-50;
L is - (C3-8 alkylene)-T-* or -(C3-8 heteroalkylene)-T-*, wherein the C3-8 alkylene or C3-8 heteroalkylene is optionally substituted with one or more oxo;
T is -CH2-, -O-, -S-, or -NH-;
-La is , wherein indicates attachment to
L and indicates attachment to Sa;
R1 is -OR1 or -NR1 -C( ())-Rr ”;
R’ is H or C1-6 alkyl;
R'' is H or C1-6 alkyl;
R1''' is C1-6 alkyl; and [0060] In some embodiments, the sialic acid lipid is of Formula (SA-II):
(SA-II) or a salt or ionized form thereof.
[0061] In some embodiments, the sialic acid lipid is of Formula (SA- IF):
(SA-II') or a salt or ionized form thereof.
[0062] n some embodiments, the sialic acid lipid is of Formula (SA-II”):
(SA-II”) or a salt or ionized form thereof. [0063] In some embodiments, the sialic acid lipid is of Formula (SA-III):
(SA-III) or a salt or ionized form thereof.
[0064] In some embodiments, the sialic acid lipid is of Formula (SA-IV):
(SA-IV) or a salt or ionized form thereof.
[0065] In some embodiments, the sialic acid lipid is of Formula (SA-V): (SA-V) or a salt or ionized form thereof.
[0066] In some embodiments, the sialic acid lipid is of Formula (SA-V-i):
(SA-V-i) or a salt or ionized form thereof.
[0067] In some embodiments, the sialic acid lipid is of Formula (SA-V-ii):
(SA-V-i) or a salt or ionized form thereof.
[0068] In some embodiments, the sialic acid lipid is of Formula (SA-VI):
(SA-VI) or a salt or ionized form thereof.
[0069] In some embodiments, the sialic acid lipid is of Formula (SA-VI-i):
(SA-VI-i) or a salt or ionized form thereof.
[0070] In some embodiments, the sialic acid lipid is of Formula (SA-VI-ii):
(SA-VI-ii) or a salt or ionized form thereof
[0071] In an aspect, the present disclosure provides a lipid comprising a diacylated propylene glycol moiety, a phosphate moiety, a PEG moiety, and a sialic acid moiety. Variables R and R ’
[0072] In some embodiments, R is C13-20 alkyl. [0073] In some embodiments, R is C15-20 alkyl. [0074] In some embodiments, R is C13-15 alkyl. [0075] In some embodiments, R is C16-18 alkyl. [0076] In come embodiments, R is C13 alkyl.
[0077] In come embodiments, R is C14 alkyl. [0078] In come embodiments, R is C15 alkyl. [0079] In some embodiments, R is C16 alkyl. [0080] In some embodiments, R is C17 alkyl. [0081] In some embodiments, R is C18 alkyl.
[0082] In some embodiments, R is C19 alkyl, [0083] In some embodiments, R is C20 alkyl. [0084] In some embodiments, R’ is C13-20 alkyl. [0085] In some embodiments, R’ is C15-20 alkyl. [0086] In some embodiments, R’ is C13-15 alkyl.
[0087] In some embodiments, R’ is C16-18 alkyl. [0088] In come embodiments, R’ is C13 alkyl. [0089] In come embodiments, R’ is C14 alkyl. [0090] In come embodiments, R’ is C15 alkyl. [0091] In some embodiments, R’ is C16 alkyl. [0092] In some embodiments, R’ is C17 alkyl.
[0093] In some embodiments, R’ is C18 alkyl. [0094] In some embodiments, R’ is C19 alkyl. [0095] In some embodiments, R1 is C20 alkyl. [0096] In some embodiments, R is C15-20 alkenyl. [0097] In some embodiments, R is C13-15 alkenyl [0098] In some embodiments, R is C16-18 alkenyl.
[0099] In come embodiments, R is C15 alkenyl. [0100] In some embodiments, R is C16 alkenyl.
[0101] In some embodiments, R is C17 alkenyl. [0102] In some embodiments, R is C18 alkenyl.
[0103] In some embodiments, R is C19 alkenyl.
[0104] In some embodiments, R is C20 alkenyl.
[0105] In some embodiments, R’ is C15-20 alkenyl.
[0106] In some embodiments, R’ is C13-15 alkenyl.
[0107] In some embodiments, R’ is C16-18 alkenyl.
[0108] In come embodiments, R’ is C15 alkenyl.
[0109] In some embodiments, R’ is C16 alkenyl.
[0110] In some embodiments, R’ is C17 alkenyl.
[0111] In some embodiments, R’ is C18 alkenyl.
[0112] In some embodiments, R’ is C19 alkenyl.
[0113] In some embodiments, R’ is C20 alkenyl.
Variables M, M’ , X+, and n
[0114] In some embodiments, n is 40 to 50.
[0115] In some embodiments, n is 41-45.
[0116] In some embodiments, n is 42-44.
[0117] In some embodiments, n is 43-44.
[0118] In some embodiments, n is 40.
[0119] In some embodiments, n is 41
[0120] In some embodiments, n is 42.
[0121] In some embodiments, n is 43.
[0122] In some embodiments, n is 44.
[0123] In some embodiments, n is 45
[0124] In some embodiments, n is 46.
[0125] In some embodiments, n is 47.
[0126] In some embodiments, n is 48.
[0127] In some embodiments, n is 49.
[0128] In some embodiments, n is 50.
[0129] In some embodiments, M is *-O-C(=O)-, wherein * indicates attachment to R.
[0130] In some embodiments, M is *-C(=O)-O-, wherein * indicates attachment to R. [0131] In some embodiments, M’ is *-O-C(=O)-, wherein * indicates attachment to R’.
[0132] In some embodiments, M’ is *-C(=O)-O-; wherein * indicates attachment to R’.
[0133] In some embodiments, X is a metal cation.
[0134] In some embodiments, X is an alkali metal cation.
[0135] In some embodiments X is a sodium cation.
[0136] In some embodiments, X is a lithium cation.
[0137] In some embodiments, X+ is a potassium cation.
[0138] In some embodiments, X+ is an ammonium cation.
Variable L and T
[0139] In some embodiments, L comprises a C3-8 alkylene moiety.
[0140] In some embodiments, L is -(C3-8 alkylene)-X’-*, wherein:
* indicates attachment to -La; wherein R is H or C1-6 alkyl; and the C1-10 alkylene is optionally substituted with one or more oxo groups.
[0141] In some embodiments, T is -CH2-.
[0142] In some embodiments, T is -O-.
[0143] In some embodiments, T is -S-.
[0144] In some embodiments, T is -NH-.
[0145] In some embodiments, the alkylene is linear.
[0146] In some embodiments, the alkylene is branched.
[0147] In some embodiments, L is --(C3-8 alkylene)-O)-*.
[0148] In some embodiments, L is -(C3-8 alkylene)-S-*.
[0149] In some embodiments, L is (C3-8 alkylene)-NH-*.
[0150] In some embodiments, L comprises a C3-8 heteroalkylene moiety.
[0151] In some embodiments, L is --(C3-8 heteroalkylene)-X’-*, wherein:
* indicates attachment to -La;
T is -CH2-, -O-, -S-, or -NR-, wherein R is H or C1-6 alkyl; and the C1 -10 alkylene is optionally substituted with one or more oxo groups. [0152] In some embodiments, the heteroalkylene is linear.
[0153] In some embodiments, the heteroalkylene is branched.
[0154] In some embodiments, L is heteroalkylene)-O-* .
[0155] In some embodiments, L is heteroalkylene)-S-*.
[0156] In some embodiments, L is heteroalkylene)-NH- * .
[0157] In some embodiments, L is , wherein indicates attachment to -La-Sa.
[0158] In some embodiments, L is
, wherein indicates attachment to -La-Sa.
[0159] In some embodiments, L is , wherein indicates attachment to -La-Sa.
[0160] In some embodiments, L is
. wherein indicates attachment to
-La-Sa.
[0161] In some embodiments, L is , wherein indicates attachment to -La-Sa.
[0162] In some embodiments, L is wherein indicates attachment to -La-Sa.
[0163] In some embodiments, L is , wherein indicates attachment to -La-Sa.
Variable La
[0164] In some embodiments. La is a lactosyl moiety is derived from lactose. In some embodiments, the lactosyl moiety is a derivative of lactose. For example, the present disclosure contemplates the use of lactose, lactosamme, or N-acetyl lactosamme, The present disclosure contemplates the use of lactosyl moieties wherein an atom from the L moiety, e.g, a heteroatom represented by X’, substitutes for a hydroxyl group in lactose. For example, the present disclosure contemplates the use of lactosyl moieties wherein the hydroxyl group at the anomeric position of lactose is replaced by a heteroatom from the L moiety. In some embodiments, the lactosyl moiety comprises lactose. In some embodiments, the lactosyl moiety comprises lactosamme. In some embodiments, the lactosyl moiety comprises N-acetyl lactosamine.
[0165] In some embodiments, La is , wherein indicates attachment to L indicates attachment to -Sa.
[0166] In some embodiments. La is . wherein indicates attachment to L indicates attachment to -Sa,
[0167] In some embodiments, La is wherein indicates attachment indicates attachment to -Sa. [0168] In some embodiments. La is , wherein indicates attachment to
L and indicates attachment to -Sa.
[0169] In some embodiments. La is
L and indicates attachment to -Sa.
[0170] In some embodiments. La is , wherein indicates attachment to L and indicates attachment to -Sa.
[0171] In some embodiments, La is , wherein indicates attachment to L and indicates attachment to -Sa. [0172] In some embodiments, La is , wherein indicates attachment to L indicates attachment to -Sa.
[0173] In some embodiments, La is , wherein indicates attachment to
L and indicates attachment to -Sa.
[0174] In some embodiments, La is , wherein indicates attachment to
L and indicates attachment to -Sa.
[0175] In some embodiments. La is , wherein indicates attachment to
L and indicates attachment to -Sa. [0176] In some embodiments, La is wherein indicates attachment to L and indicates attachment to -Sa.
Variable Sa
[0177] In some embodiments, Sa is a sialic acid moiety comprising neuraminic acid. In some embodiments, the sialic acid comprises a derivative of neuraminic acid. In some embodiments, the sialic acid comprises N-glycolneuramimc acid. In some embodiments, the sialic acid comprises 2- keto-3-deoxynonic acid. In some embodiments, the sialic acid comprises N-acylated neuraminic acid. In some embodiments, Sa is indicates attachment to -
La, In some embodiments, Sa is wherein indicates attachment to the lactosyl moiety.
Variable -La-Sa
[0178] In some embodiments, -La-Sa is
[0179] In some embodiments, -La-Sa is
[0180] In some embodiments, -La-Sa is
[0181] In some embodiments, -La-Sa is
[0182] In some embodiments, -La-Sa is [0183] In some embodiments, -La-Sa is
[0184] In some embodiments, -La-Sa is
[0185] In some embodiments, -La-Sa is
[0186] In some embodiments, -La-Sa is
[0187] In some embodiments, -La-Sa is
[0188] In some embodiments, -La-Sa is
[0189] In some embodiments, -La-Sa is
Exemplary Embodiments
[0190] In some embodiments, the sialic acid lipid is selected from the compounds described in Table SA-1, salts thereof, and ionized forms thereof.
[0191] In some embodiments, the sialic acid lipid is selected from the compounds described in Table SA-1 and salts thereof
[0192] In some embodiments, the sialic acid lipid is selected from the compounds described in Table SA-1, and ionized forms thereof.
[0193] In some embodiments, the sialic acid lipid is selected from the compounds described in Table SA-1.
to
\D
Lipid Manoparticles of the Present Disclosure
[0194] In some aspects, the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid disclosed herein.
[0195] In some embodiments, the lipid nanoparticle further comprises an ionizable lipid.
[0196] In some embodiments, the lipid nanoparticle further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof.
[0197] In some embodiments, the lipid nanoparticle comprises a sialic acid lipid disclosed herein, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
[0198] In some aspects, the present disclosure provides a population of lipid nanoparticles comprising a sialic acid lipid disclosed herein.
[0199] In some embodiments, the population of lipid nanoparticles further comprises an ionizable lipid.
[0200] In some embodiments, the population of lipid nanoparticles further comprises a phospholipid, a PEG lipid, a structural lipid, or any combination thereof.
[0201] In some embodiments, the population of lipid nanoparticles comprises a sialic acid lipid disclosed herein, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
[0202] The present disclosure provides lipid nanoparticles and populations of lipid nanoparticles comprising a sialic acid lipid, an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid.
[0203] Without wishing to be bound by theory, the LNPs of the present disclosure may facilitate delivery of nucleic acid payloads to certain cell types or when expression of a protein of interest is desired (e.g., a polypeptide to which an immune response is desired or polypeptide for therapeutic expression) to improve the quality of immune response to an antigen or to dampen the immune response to a therapeutic. For example, the LNPs disclosed herein that, incorporate Compound No. 9 can lead to reduced Th2 cytokine production (IL-4, IL-5 and IL- 15) while maintaining Thl cytokine production (IFNy, TNFa and 11,-2) and CD4+ and CD8+ T cell activation.
[0204] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to human CD33+ hematopoietic stem cells.
[0205] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to CD169+ marginal Macrophages.
[0206] In some embodiments, LNPs comprising Compound No. 9 are suitable for delivery of nucleic acids polypeptides to promote an immune response (e.g., an antigen as described herein). In some embodiments, the LNPs comprising Compound No. 9 improve the quality of the immune response. For example, LNPs comprising Compound. No. 9 can improve the quality of an immune response to a. vaccine.
[0207] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a therapeutic polypeptide when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 1 show reduced immune response compared to LNPs that do not comprise Compound No. 1.
[0208] In some embodiments, LNPs comprising Compound No. 9 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 9 show' reduced immune response compared to LNPs that do not comprise Compound No. 9.
[0209] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to myeloid progenitor cells, e.g. 11CD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
[0210] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a. therapeutic polypeptide to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
[0211] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a therapeutic polypeptide to hematopoietic stem and progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
[0212] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery' of a nucleic acid encoding a. therapeutic polypeptide to erythroid progenitor cells.
[0213] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
[0214] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to erythroid progenitor cells.
[0215] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acid encoding CRISPR protein and/or gRNA to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells. [0216] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery' of one or more nucleic acids encoding a CRISPR protein and/or gRNA to hematopoietic stem and. progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
[0217] Without wishing to be bound by theory, it is understood that when administered to subjects, the LNPs comprising sialic acid lipids may result in reduced cytokine secretion, reduced inflammatory responses, increased targeting to bone marrow resident HSPCs, differential targeting of myeloid subsets, and enhanced protein production in the liver, as compared to LNPs of different composition.
[0218] Without, wishing to be bound by theory, it is understood that LNPs comprising sialic acid lipids formulated with sialic acid lipid added in the lipid stock solution may generally be small in size.
[0219] Without wishing to be bound by theory , it is understood that the more-anionic zeta potentials observed for LNPs comprising sialic acid lipids relative to LNPs that lack sialic acid lipids may be due to the presence of negatively charged sialic acid moieties on the surface.
[0220] Without washing to be bound by theory, it is understood that LNPs that the LNPs comprising sialic acid lipids may bind to human CD-33 more strongly than LNPs of different formulation.
[0221] Without wishing to be bound by theory, it i s understood that the LNPs comprising sialic acid lipids may effectuate protein expression in HSPCs (e.g., Lin-cKit+Scal+ cells).
[0222] Without wishing to be bound by theory, it is understood that the LNPs comprising sialic acid lipids may effectuate protein expression in Lin-cKit+Scal- cells (LK cells).
[0223] Without wishing to be bound, by theory, it is understood that the LNPs comprising sialic acid lipids may effectuate protein expression in erythroid progenitor cells.
[0224] Without wishing to be bound by theory , it is understood that the LNPs comprising sialic acid lipids may effectuate protein expression in long-term HSCs (LT-HSCs, e.g., Lin- cKit+Scal+CD150+CD48-cells).
[0225] Without wishing to be bound by theory , it is understood that the process by which the LNPs comprising sialic acid lipids are made may play a role in the ability of the LNPs to deliver mRNAs to bone marrow.
[0226] Without wishing to be bound by theory, it is understood that the LNPs comprising sialic acid, lipids may effectuate lower Seal expression (e.g., no significant Seal expression) relative to the Seal expression effectuate by LNPs of different composition.
[0227] Without wishing to be bound by theory , it is understood that LNPs comprising sialic acid lipids may efficiently target cells such as HSPCs without activating an immune response, e.g., an inflammatory response measured by way of Seal expression, within said cells. It is believed that LNPs of alternative composition that are equivalently efficient at targeting said cells would, in contrast to LNPs comprising sialic acid lipids, activate an immune response, e.g., an inflammatory response measured by way of Seal expression, within said cells.
[0228] In some embodiments, the present disclosure provides a lipid nanoparticle comprising a sialic acid lipid, an ionizable lipid, and a structural lipid, wherein the sialic acid lipid is of Formula (SA-I).
[0229] In some embodiments, a phospholipid useful or potentially useful in the present invention is an anionic phospholipid.
[0230] In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 5 mol %, about 0.1 mol % to about 4 mol %, about 0.1 mol % to about 3 mol %, about 0.1 mol % to about 2 mol %, about 0.2 mol % to about 2 mol %, about 0.4 mol % to about 1.5 mol % of the sialic acid lipid, or about 0.4 mol% to about 1 mol% of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0. 1 mol % to about 5 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 4 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 3 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.1 mol % to about 2 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.2 mol % to about 2 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.4 mol % to about 1.5 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises between about 0.4 mol % to about 1 mol % of the sialic acid lipid.
[0231] In some embodiments, the population of lipid nanoparticles comprises about 0.1 mol %, about 0.2 mol %, about 0.3 mol %, about 0.4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, or about 2.0 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.5 mol % of the sialic acid, lipid. In some embodiments, the population of lipid, nanoparticles comprises about 0.6 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.7 mol % of the sialic acid lipid. In some embodiments, the population of lipid, nanoparticles comprises about 0.8 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.9 mol % of the sialic acid lipid. In some embodiments, the population of lipid nanoparticles comprises about 1.0 mol % of the sialic acid lipid.
[0232] In some embodiments, the ionizable lipid is compound 1-301, compound II-6, 1-25, or 1-18. In some embodiments, the ionizable lipid is compound 1-301 or compound II-6,
[0233] In some embodiments, the ionizable lipid is compound 1-301. Compound 1-301 is a compound of the formula. or a salt thereof.
[0234] In some embodiments, the ionizable lipid is compound II-6. Compound II-6 is a compound of the formula or a salt thereof.
[0235] In some embodiments, the ionizable lipid is 1-18. 1-18 is a compound of the formula or a salt thereof.
[0236] In some embodiments, the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of the ionizable lipid. In some embodiments, the population of lipid nanoparticles comprises about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, or about 50 mol % of the ionizable lipid. In some embodiments, the population of lipid nanoparticles comprises about 30 mol % to about 50 mol % of the ionizable lipid (e.g., compound 1-301, compound 1-18, or compound II-6). In some embodiments, the population of lipid nanoparticles comprises about 35 mol % to about 45 mol % of the ionizable lipid (e.g., compound 1-301, compound 1-18, or compound II-6).
[0237] In some embodiments, the population of lipid, nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound 301. In some embodiments, the population of lipid nanoparticles comprises about. 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 47 mol %, or about 50 mol % of compound 1-301.
[0238] In some embodiments, the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound 301 . In some embodiments, the population of lipid, nanoparticles comprises about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 47 mol %, or about 50 mol % of compound 1-18.
[0239] In some embodiments, the population of lipid nanoparticles comprises about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of compound II-6. In some embodiments, the population of lipid nanoparticles comprises about 30 mol %, about 35 mol %, about. 40 mol %, about 45 mol %, about 47 mol %, or about. 50 mol % of compound II-6.
[0240] In some embodiments, the structural lipid is cholesterol.
[0241] In some embodiments, the population of lipid nanoparticles comprises about 15 mol % to about 50 mol %, about 20 mol % to about 50 mol %, about 25 mol % to about 50 mol %, about. 30 mol % to about 50 mol %, about 35 mol % to about 50 mol %, about 40 mol % to about 50 mol %, or about 45 mol % to about 50 mol % of the structural lipid (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 15 mol % to about 45 mol %, about 20 mol % to about 45 mol %, about 25 mol % to about 45 mol %, about 30 mol % to about 45 mol %, about 35 mol % to about 45 mol %, or about 40 mol % to about 45 mol % of the structural lipid (e.g., cholesterol). In some embodiments the population of lipid nanoparticles comprises about 15 mol % to about 40 mol %, about 20 mol % to about 40 mol %, about 25 mol % to about 40 mol %, about 30 mol % to about 40 mol %, or about 35 mol % to about 40 mol % of the structural lipid, (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 20 mol % to about 45 mol % of the structural lipid (e.g;, cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 20 mol % to about 40 mol % of the structural lipid (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 30 mol % to about 40 mol % of the structural lipid, (e.g., cholesterol).
[0242] In some embodiments, the population of lipid nanoparticles comprises about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 39 mol%, about 40 mol %, about. 45 mol %, or about 50 mol % of the structural lipid (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 35 mol % of the structural lipid (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 40 mol % of the structural lipid (e.g., cholesterol). In some embodiments, the population of lipid nanoparticles comprises about 45 mol % of the structural lipid (e.g., cholesterol).
[0243] In some embodiments, the population of lipid nanoparticles comprises a phospholipid. In some embodiments, it comprises about 10 mol % to about 30 mol %, about 15 mol % to about 25 mol %, or about 15 mol % to about 20 mol % of the phospholipid. In some embodiments, it comprises about 10 mol %, about 15 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 25 mol %, or about 30 mol % of the phospholipid. In some embodiments, the phospholipid is DMPS , DSPC, DOPE, DOPC, POPE, or POPC.
[0244] In some embodiments, the population of lipid nanoparticles comprises a phospholipid that is DSPC, wherein the population of lipid nanoparticles comprises about 10 mol % to about 30 mol %, about. 10 mol % to about 25 mol %, about 10 mol % to about 20 mol %, about. 15 mol % to about 25 mol %, or about 15 mol % to about 20 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 10 mol % to about 25 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 10 mol % to about 20 mol % of the DSPC. In some embodiments, the population of lipid, nanoparticles comprises about 15 mol % to about 25 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 15 mol % to about 20 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprise about 10 mol % to about 15 mol % of the DSPC.
[0245] In some embodiments, the population of lipid nanoparticles comprises a phospholipid that is DSPC, wherein the population of lipid nanoparticles comprises about 10 mol %, about 11 mol %, about 15 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 25 mol %, or about 30 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 10 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 20 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 22 mol % of the DSPC. In some embodiments, the population of lipid nanoparticles comprises about 25 mol % of the DSPC.
[0246] In some embodiments, the population of lipid nanoparticles is free of PEG- lipid.
[0247] In some embodiments, the population of lipid nanoparticles further comprises a PEG lipid. In some embodiments, the population of lipid nanoparticles comprises about 0.5 mol % to about 10 mol %, about 0.5 mol % to about 5 mol %, about 0.5 mol% to about 3 mol%, about 1 mol % to about 5 mol %, or about 1 mol % to about 3 mol % of the PEG lipid.
[0248] In some embodiments, the PEG lipid is PL-02. PL-02 refers to a polymer of the formula: or a salt thereof, wherein r is 45. As one of ordinary skill in the art would understand, the number of repeating units indicated, in the structure of a polymer refers to the average number of repeating units (a.k.a., average degree of polymerization). E.g., in some embodiments, r is an integer from about 35 to about 55.
[0249] In some embodiments, the population of lipid nanoparticles comprises about 1 mol % to about 5 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about 3 mol % to about 5 mol % of the PEG lipid (e.g., PL-02).
[0250] In some embodiments, the population of lipid nanoparticles comprises about 0.5 mol %, about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, or about 5 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about. 3 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about 4 mol % of the PEG lipid (e.g., PL-02). In some embodiments, the population of lipid nanoparticles comprises about 5 mol % of the PEG lipid (e.g., PL-02). [0251] In some embodiments, the population of lipid nanoparticles has a. pH value lower than the pKa value of the ionizable lipid. In some embodiments, it has a pH value of about 4.0+2.0, about 4.0+1.5, about 4.0+1.4, about 4.0+ 1.3, about 4.0+1.2, about 4.0+1.1, about 4.0+1.0, about 4.0+0.9, about 4.0+0.8, about 4.0+0.7, about 4.0+0.6, about 4.0+0.5, about 4.0+0.4, about 4.0+0,3, about 4.0+0.2, or about 4.0+0.1. In some embodiments, it has a pH value of about 5.0+2.0, about 5.0+1.5, about 5.0+1.4, about 5.0+1.3, about 5.0+1.2, about 5.0+1. 1, about 5.0+1.0, about 5.0+0.9, about 5.0+0.8, about 5.0+0.7, about 5.0+0.6, about 5.0+0.5, about 5.0+0.4, about 5.0+0.3, about 5.0+0.2, or about 5.0+0.1.
[0252] In some embodiments, the population of lipid nanoparticles has a pH value higher than the pKa value of the ionizable lipid. In some embodiments, it has a pH value of about 8.0+2.0, about 8.0+1.5, about 8.0+1.4, about 8.0+1.3, about 8.0±1.2, about 8.0+1 .1 , about 8.0+1 .0, about 8.0+0.9, about 8.0+0.8, about 8.0+0.7, about 8.0+0.6, about 8.0+0.5, about 8.0+0.4, about 8.0+0.3, about 8.0+0.2, or about 8.0+0.1. In some embodiments, the population of lipid nanoparticles has a pH value of about 9.0+3.0, about 9.0+2.0, about 9.0+1.5, about 9.0+1.4, about 9.0+1.3, about 9.0+1.2, about 9.0+1.1 , about. 9.0+1 .0, about 9.0+0.9, about 9.0+0.8, about 9.0+0.7, about 9.0+0.6, about 9.0+0.5, about 9.0+0.4, about 9.0+0.3, about 9.0+0.2, or about 9.0+0. 1. In some embodiments, the population of lipid nanoparticles has a pH value of about 12.0+2.0, about 12.0+1.5, about 12.0+1.4, about 12.0+1.3, about 12.0+1.2, about 12.0+1.1, about 12.0+1.0, about 12.0+0.9, about. 12.0+0.8, about 12.0+0.7, about 12.0+0.6, about 12.0+0.5, about 12.0+0.4, about 12.0+0.3, about 12.0+0.2, or about 12.0+0.1.
[0253] In some embodiments, the population of lipid nanoparticles has a zeta potential between about -40m V to about - 1 mV, about -40m V to about -5m V, about -30mV to about -5m V, about -20m V to about -5m V, about -40m V to about -10mV, about -30mV to about -10mv, or about -20m V to about -10m V when measured in 0.1N PBS at pH 7.5. In some embodiments, the population of lipid nanoparticles has a zeta potential between about -20m V to about -10m V when measured in 0.1N PBS at pH 7.5.
[0254] In some embodiments, the population of lipid nanoparticles is free of therapeutic agent. [0255] In some embodiments, the population of lipid nanoparticles further comprises a therapeutic agent. In some embodiments, the therapeutic agent is an mRNA.
[0256] Embodiments of the present disclosure are directed to pharmaceutical compositions comprising the population of lipid nanoparticles described herein and one or more pharmaceutically acceptable carriers or excipients. Some embodiments are directed, to methods of preparing the pharmaceutical compositions.
[0257] Embodiments of the present disclosure are directed to a method of preparing the population of lipid nanoparticles as described herein.
[0258] Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
[0259] Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described, herein for use in delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject.
[0260] Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject.
[0261] Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
[0262] Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to erythroid progenitor cells (EPC) in a subject.
[0263] Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to an erythroid progenitor cells (EPC) in a subject.
[0264] Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a myeloid cell in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
[0265] Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to myeloid cells in a subject.
[0266] Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a therapeutic agent to myeloid cells in a subject.
[0267] Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a. therapeutic agent to myeloid cells in a subject.
[0268] Embodiments of the present disclosure are directed to methods of delivering a therapeutic agent to a lymphoid cell in a subject, comprising administering to the subject the population of lipid nanoparticles or the pharmaceutical compositions as described herein.
[0269] Embodiments of the present disclosure are directed to a population of lipid nanoparticles or pharmaceutical compositions as described herein for use in delivering a therapeutic agent to lymphoid cells in a subject.
[0270] Embodiments of the present disclosure are directed to use of the population of lipid nanoparticles or the pharmaceutical compositions as described herein in the manufacture of a medicament for delivering a therapeutic agent to lymphoid cells in a subject. [0271] In addition to targeting desired cell populations, the subject LNPs can be used to deliver nucleic acid molecules encoding polypeptides against which an immune response is not desired. The reduced levels of cytokine production in subjects to which these LNPs are administered make them particularly desirable delivery vehicles, e.g., for therapeutic protein expression.
[0272] In some embodiments, the subject is human.
[0273] In some embodiments, an empty lipid nanoparticle solution may be prepared by Process 1, Process 1 comprising: i) a nanoprecipitation step, comprising: i-a) a mixing step, comprising mixing a lipid solution comprising an ionizable lipid, a structural lipid, and a phospholipid, with a first aqueous buffer solution, thereby forming an intermediate empty-iipid nanoparticle solution (intermediate empty-LNP solution) comprising an intermediate empty lipid nanoparticle (intermediate empty LNP), i-b) a holding step, comprising holding the intermediate empty-LNP solution for a residence time, and i-c) a diluting step, comprising adding a diluting solution comprising a second aqueous buffer solution to the intermediate empty-LNP solution, thereby forming the empty-LNP solution comprising an empty LNP, wherein the lipid solution, the aqueous buffer solution, and/or the diluting solution comprises a. phosphatidylserine phospholipid.
[0274] In some embodiments, a loaded lipid nanoparticle solution may be prepared by Process 1, Process 1 further comprising:
1) a nanoprecipitation step, comprising: i-a) a mixing step, comprising mixing a lipid solution comprising an ionizable lipid, a structural lipid, and a phospholipid, with a first aqueous buffer solution, thereby forming an intermediate empty-iipid nanoparticle solution (intermediate empty-LNP solution) comprising an intermediate empty lipid nanoparticle (intermediate empty LNP): i-b) a holding step, comprising holding the intermediate empty-LNP solution for a residence time; and i-c) a diluting step, comprising adding a diluting solution comprising a second aqueous buffer solution to the intermediate empty-LNP solution, thereby forming the empty-LNP solution comprising an empty LNP, iii) mixing a nucleic acid solution comprising a nucleic acid with the empty-LNP solution, thereby forming the loaded-LNP solution comprising a loaded lipid nanoparticle (loaded LNP), wherein the lipid solution, the aqueous buffer solution, and/or the diluting solution comprises a phosphatidylserine phospholipid.
[0275] In some embodiments of Process 1, the lipid solution comprises the phosphatidylserine phospholipid.
[0276] In some embodiments of Process 1, the aqueous buffer solution comprises the phosphatidylserine phospholipid.
[0277] In some embodiments of Process 1, the diluting solution comprises the phosphatidylserine phospholipid.
[0278] In some embodiments of Process 1 , the empty LNP comprises the phosphatidylserine phospholipid.
[0279] In some embodiments of Process 1, the mixing step is performed with a first aqueous buffer solution having a pH higher than the pKa of the ionizable lipid.
[0280] In some embodiments, the mixing step is performed at a pH of 4.0 to 12.0.
[0281] In some embodiments, the mixing step is performed at a pH of 12.0±2.0, 12.0 : 1 .5, 12.0±1.0, 12.0±0.9, 12.0±-0.8, 12.0±0.7, 12.0±0.6, I2.0±0.5, 12.0±0.4, 12.0±0.3, 12.0±0.2, or 12.0±0.1.
[0282] In some embodiments of Process 1, the lipid solution comprises one or more phosphatidylserine phospholipid.
[0283] In some embodiments of Process 1, the phosphatidylserine phospholipid, is DSPC, DMPS, or a mixture thereof.
[0284] In some embodiments of Process 1, the pH value of the diluting solution is about 12.0±3 0, 12.0±2.0, 11.0±1.5, 11.0±1.0 , 11.0±0.9, 11.0±0.8 , 11.0±0.7, 11.0±0.6, 11.0±0 5. 1100±0.4, 11.0± 0.3, 11.0±0.2, or 11.0±0.1 (e.g., about 11.6).
[0285] In some embodiments of Process 1, the pH value of diluting solution is about 5.0±-2.0 , 5.0±1.5, 5.0±1.0, 5.0±0.9, 5.0±0.8, 5.0±0.7, 5.0±0.6, 5.0±0.5, 5.0±0.4, 5.0±0.3, 5.0±0.2, or 5.0±0.1 (e.g., about 4.4).
Phospholipids
[0286] Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a. phospholipid moiety and one or more fatty acid moieties. [0287] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
[0288] A fatty acid, moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
[0289] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g., delivery of one or more elements to a. target tissue.
[0290] Particular phospholipids can facilitate cellular uptake and/or fusion. For example, an anionic phospholipid (e.g., phospholipids comprising phosphatidyl glycerol or phosphatidylserine) can interact with one or more receptors of a cell (e.g. , receptors of a cellular or intracellular membrane). Cellular uptake of a lipid-containing composition (e.g, LNPs) can allow for subcellular trafficking of the lipid-containing composition (e.g, entry into endosomes or transport to the endoplasmic reticulum). Anionic phospholipids can also facilitate fusion to a membrane. Interaction of an anionic phospholipid with a receptor of a cell can bring elements of a lipid-containing composition into contact with a membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g, a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g, delivery of one or more elements to a target tissue.
[0291] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g, an alkenyl group in which one or more double bonds are replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a. useful component such as a targeting or imaging moiety (e.g., a dye). [0292] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidyl glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipids, such as sphingomyelin.
[0293] In other embodiments, a therapeutic and/or prophylactic is a protein, for example, a protein needed to augment or replace a naturally occurring protein of interest. Such proteins or polypeptides may be naturally occurring, or may be modified using methods known in the art, e.g., to increase half-life. Exemplary proteins are intracellular, transmembrane, or secreted.
Ionizable Lipids
[0294] In some embodiments, a lipid nanoparticle (e.g., an empty LNP or a loaded LNP) of the disclosure further includes one or more ionizable lipids in addition to the sialic acid lipid of Formula (SA-I).
[0295] In some embodiments, the ionizable lipid is of compound of Formula (IL-A): (IL-A) or its N -oxide, or a salt or isomer thereof, wherein:
R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
R2 and RJ are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*0R”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, -(CH2)nQ, and unsubstituted C1- 6 alkyl, where Q is selected from a carbocycle, heterocycle, and and , wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S; and a is
1, 2, 3, or 4; wherein denotes a point of attachment; each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5;
R8 is selected from the group consisting of C3-6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO2, Ci-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle;
R12 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; each R is independently selected from the group consisting of C1-6 alkyl, C1-3 alkyl-aryl, C2-3 alkenyl, and H;
RA is selected from H and C1-3 alkyl;
RSX is selected from a C3-8 carbocycle, a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, C1-6 alkyl, C2-6 alkenyl, (C1-3 alkoxy )C1-3 alkyl, (CH2)p1O(CH2)P2RSX1, and (CH2)p1RSX1, wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C1-6 alkyl, and (C1-3 alkoxy)C1-3 alkyl;
RSX1 is selected from C(O)NR14R14’, a C3-8 carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C1-3 alkyl, (C1-3 alkoxy )C1-3 alkyl, C1-6 alkylamino, di-(C1-6 alkyl) amino, and NH2; each R13 is selected from the group consisting of OH, oxo, halo, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C1-6 alkylamino, di-(C1-6 alkyl) amino, NH2, C(O)NH2, CN, and NO2;
R14 and R’4 are each independently selected from the group consisting of H and C1-6 alkyl; p1 is selected from 1, 2, 3, 4, and 5; p2 is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each Rb is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
M and M’ are independently selected from an aryl group, and a heteroaryl group, in which M” is a bond, C1-13 alkyl or C2- 13 alkenyl; each RM is independently selected from the group consisting of H, C1-6 alkyl and
C2-6 alkenyl; each R’ is independently selected from the group consisting of C1 -18 alkyl, C2-18 alkenyl. -R*YR’; -YR”, (CH2)q-OR*, and H, and each q’ is independently selected, from 1, 2, and 3; each R” is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle, each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 1 1 , 12, and 13.
^0296^ In some embodiments, the ionizable lipid is of compound of Formula (IL-B): (IL-B) or its N-oxide, or a. salt or isomer thereof, wherein R’a is R branched. branched - s. ; wherein denotes a point of attachment; wherein Raα, Raβ, Raγ, andRaδ are each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;
R2 and RJ are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R4 is selected from the group consisting of , wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and wherein denotes a point of attachment; wherein
R10 is N(R)2, each R is independently selected from the group consisting of C1 - 6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl,
C2-3 alkenyl, and H;
M: and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;
R’ is a C1-12 alkyl or C2-12 alkenyl;
1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.
[0297] In some embodiments, the ionizable lipid is of compound a compound of Formula (IL- C): a salt or isomer thereof, wherein
1 is selected from 1, 2, 3, 4, and 5;
M1 is M' ;
R4 is -(CH2)nQ, in which Q is OH, and n is selected from 1, 2, 3, 4, or 5;
M and M’ are independently selected from -C(O)O- and -OC(O)-;
R2 and R3 are both C1 -14 alkyl, or C2-14 alkenyl; and
R’ is a C1-C12 linear alkvl.
[0298] In some embodiments, the ionizable lipid is of compound a compound of Formula (II
D): (IL-D) or its N-oxide, or a salt or isomer thereof, wherein R,a is or R wh erein i s: and R’b is: wherein denotes a point of attachment; wherein Raγ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
R2 and RJ are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;
R4 is wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;
R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
[0299] In some embodiments, the ionizable lipid is a. of compound of Formula. (IL-I):
(IL-I) or its N-oxide, or a salt or isomer thereof, wherein:
R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
R2 and R3 are independently selected from the group consisting of H, C1 -14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;
R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, , and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, , each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of OH, C1-3 alkyl, C2-3 alkenyl, and H;
M and M’ are independently selected from ( ) ( ) ( ) S(O)2-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C1 -13 alkyl or C2- 13 alkenyl;
R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
R8 is selected, from the group consisting of C3-6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle;
R10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, (CH2)qOR*, and H, and each q is independently selected from 1, 2, and 3; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-15 alkyl and
C3-15 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and
C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.
[0300] In some embodiments, the ionizable lipid is a of compound, of Formula (IL-IA): or a salt or isomer thereof, wherein 1 is selected from 1 , 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;
M1 is a bond or M’;
R4 is un substituted C1-3 alkyl, or -(CH2)nQ, in which Q is or heteroaryl, and each n is selected from 1, 2, 3, 4, or 5;
M and M’ are independently selected from , art aryl group, and a heteroaryl group; and
R2 and R3 are both C1-14 alkyl or C2-14 alkenyl;
R8 is selected from the group consisting of C3-6 carbocycle and heterocycle;
R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; and
R’ is a C1 -20 alkyl or C2-18 alkenyl.
[0301] In some embodiments, the ionizable lipid is a. compound of Formula (IL-IB): (IL-IB), or its N-oxide, or a salt or isomer thereof, wherein
1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9;
R is selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; and
R2 and R3 are independently selected from the group consisting of C1-14 alkyl, and C2- 14 alkenyl;
M and M’ are independently selected from -C(O)O- and -OC(O)-;
RN is H, or C1-3 alkyl;
Xa and Xb are each independently O or S;
R10 is selected from the group consisting of H, halo, heteroaryl, a carbocycle, a. heterocycle, aryl and heteroaryl; each R is independently selected from the group consisting of C1-12 alkyl, C2-12 alkenyl, and H; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; r is 0 or 1; t1 is selected from 1, 2, 3, 4, and 5; p1 is selected from 1, 2, 3, 4, and 5; q1 is selected from 1, 2, 3, 4, and 5; and s1 is selected from 1, 2, 3, 4.
[0302] In some embodiments, the ionizable lipid is a compound of Formula (IL-IC): (IL-IC), or its N-oxide, or a salt or isomer thereof, wherein R’a is or ; wherein is and is: ; and
R,b is: wherein denotes a point of attachment; wherein Raγ, Raγ and Ray are each C1-12 alkyl or C2-12 alkenyl;
Rbγ is H, C1 -12 alkyl or C2-12 alkenyl;
R2 and R3 are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;
R4 is -(CH2)nOH; or wherein denotes a point of attachment; each R’ independently is a C1-12 alkyl or C2-12 alkenyl;
R10 is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n and n2 are each selected from the group consisting of 1 , 2, 3, 4, and 5;
Ya is a C3-6 carbocycle;
R*”a is selected from the group consisting of C1-15 alkyl and C2-15 alkenyl;
1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; and s i s 2 or 3. [0303] In some embodiments, the ionizable lipid is a compound of Formula (IL*) or a salt thereof, wherein:
R1 is -OH, -NRN-C4-10 cycloalkenyl optionally substituted with one or more oxo or
RN IS H or C1-6 alkyl;
R is H or C1-6 alkyl;
RN’ is H or C1-6 alkyl, o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8, m is 4, 5, 6, 7, or 8;
M is - wherein * indicates attachment to R2;
M’ is wherein * indicates attachment to R3;
R2 is or -(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl;
R2a is -H or C1-10 alkyl;
R2b is -H or C1-10 alkyl;
R2C is C1-8 alkyl or C2-8 alkenyl; R3 is
R3a is H or C1-10 alkyl;
R3b is H ar C1-8 alkyl; and
Rto is C1-10 alkyl or C2-8 alkenyl.
[0304] In some embodiments, the ionizable lipid is of Formula (IL**-I): or a salt thereof, wherein :
R1 is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8;
M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;
M’ is -C(=O)-O-*, wherein * indicates attachment to R3;
R2C is C4-8 alkyl;
R3a is C7-10 alkyl; and.
R3C is C3-5 alkyl.
[0305] In some embodiments, the ionizable lipid is of Formula (IL**-III): or a salt thereof, wherein : R1 is NRN- C4-10 cycloalkenyl optionally substituted with one or more oxo or -
RN is H;
R is C1-2 alkyl;
RN is H, o is 2, 3, or 4; n is 6, 7, or 8;
M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;
M/ is -C(=O)-O-*, wherein * indicates attachment to R3;
R2a is C7-10 alkyl;
R2C is C4-6 alkyl;
R3a is C1-3 alkyl; and
Rto is C4-6 alkyl.
[0306] In some embodiments, the ionizable lipid is of Formula (IL**-IV): or a salt thereof, wherein:
R1 is OH; o is 2, 3, or 4; n is 6, 7, or 8;
M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;
M’ is -C(=O)-O-*, wherein * indicates attachment to R3;
R2b is C3-5 alkyl;
R2C is C2-4 alkyl;
R3a is C7-10 alkyl; and.
Rto is C4-6 alkyl.
[0307] In some embodiments, the ionizable lipid is of Formula (IL*-I); or a salt thereof, wherein:
R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*; and
R3a is C1-8 alkyl.
[0308] In some embodiments, ionizable lipid is of Formula (IL*-Ia): or a salt thereof, wherein:
R1, o, m, n, M, M’, R2c, and R3c are as defined for Formula IL*; and
R3a is C1-8 alkyl.
[0309] In some embodiments, the ionizable lipid is of Formula. (IL*-Ia’): or a salt thereof, wherein: o, , and R3c are as defined for variable IL*, and
R3a is C1-8 alkyl.
[0310] In some embodiments, the ionizable lipid is of Formula. (IL *-IIa): or a salt, thereof, wherein:
R1, o, m, n, M, M’, R2c, and R3c are as defined for Formula IL*; and R3a is C1-8 alkyl.
[0311] In some embodiments, the ionizable lipid is of Formula (IL*-II'): or a salt thereof, wherein: o, M, XL, R2C and R3c are as defined for variable IL*; and
R3a is C1-8 alkyl.
[0312] In some embodiments, the ionizable lipid is of Formula (IL*-III): or a salt thereof, wherein:
R3, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*;
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
[0313] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein:
R3, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*;
R2b is a C1-8 alkyl; and.
R3a is C1-8 alkyl.
[0314] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein:
R1, o, M, M’, R2C, and R3c are as defined for variable IL*;
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
[0315] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’): or a salt thereof, wherein:
R1, o, M, M\ R2C, and R3c are as defined for variable IL*;
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
[0316] In some embodiments, the ionizable lipid is of Formula (ILMIIb): or a salt thereof, wherein;
R1, o, M, M', R2c, and R3c are as defined for variable IL*,
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
[0317] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’): or a salt thereof, wherein;
R3, o, M, M’, R2c, and R3c are as defined for variable IL*;
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
[0318] In some embodiments, the ionizable lipid is of Formula. (IL*-IV): or a salt thereof, wherein;
R1, o, m, n, M, M’, R2c and R3c are as defined for variable IL*;
R2b is a. C1-8 alkyl, and
R3a is C1-8 alkyl.
[0319] In some embodiments, the ionizable lipid is of Formula (IL*-IVa): or a salt thereof, wherein;
R1, o, m, n, M, M’, R2c, and R3c are as defined for variable IL*;
R2b is a C1-8 alkyl; and
R3 a is C1-8 alkyl.
[0320] In some embodiments, the ionizable lipid is of Formula (JL*-Iva’): or a salt thereof, wherein: o, M, M’, R2c, and R3c are as defined for variable IL*,
R2a is a C1-8 alkyl; and
R3a is C1-8 alkyl.
Variables o, of Ionizable Lipid
[0321] In some embodiments of the ionizable lipid, o is 1.
[0322] In some embodiments of the ionizable lipid, o is 2.
[0323] In some embodiments of the ionizable lipid, o is 3.
[0324] In some embodiments of the ionizable lipid, o is 4.
[0325] In some embodiments of the ionizable lipid, R1 is -OH.
[0326] In some embodiments of the ionizable lipid, RN is H.
[0327] In some embodiments of the ionizable lipid, RN is methyl.
[0328] In some embodiments of the ionizable lipid, RN is ethyl.
[0329] In some embodiments of the ionizable lipid, R1 is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RN’RN ’).
[0330] In some embodiments of the ionizable lipid, RN is H.
[0331] In some embodiments of the ionizable lipid, RN is methyl.
[0332] In some embodiments of the ionizable lipid, RN’ is ethyl.
[0333] In some embodiments of the ionizable lipid, R^” is II
[0334] In some embodiments of the ionizable lipid, RN is methyl.
[0335] In some embodiments of the ionizable lipid, RN is ethyl.
[0336] In some embodiments of the ionizable lipid, RN is H and RN is methyl.
[0337] In some embodiments of the ionizable lipid, R1 is [0338] In some embodiments of the ionizable lipid. R1 is
Variables m and n of the Ionizable Lipid.
[0339] In some embodiments of the ionizable lipid, m is 4.
[0340] In some embodiments of the ionizable lipid, m is 5.
[0341] In some embodiments of the ionizable lipid, m is 6.
[0342] In some embodiments of the ionizable lipid, m is 7.
[0343] In some embodiments of the ionizable lipid, m is 8.
[0344] In some embodiments of the ionizable lipid, m is 4.
[0345] In some embodiments of the ionizable lipid, n is 5.
[0346] In some embodiments of the ionizable lipid, n is 6.
[0347] In some embodiments of the ionizable lipid, n is 7.
[0348] In some embodiments of the ionizable lipid, n is 8.
[0349] In some embodiments of the ionizable lipid, n is 5 and m is 7.
[0350] In some embodiments of the ionizable lipid, n is 7 and m is 7.
[0351] In some embodiments of the ionizable lipid, m is 6 and n is 6.
Variables M andM’
[0352] In some embodiments of the ionizable lipid, M is wherein * indicates attachment to R2.
[0353] In some embodiments of the ionizable lipid, M is wherein * indicates attachment to R2.
[0354] In some embodiments of the ionizable lipid, M’ is wherein * indicates attachment to R3.
[0355] In some embodiments of the ionizable lipid, M’ is wherein * indicates attachment to R3.
[0356] In some embodiments of the ionizable lipid, M is wherein * indicates attachment to R2, and M’ is wherein * indicates attachment to R3
Variables R2, R2a, R2b, R2c [0357] In some embodiments of the ionizable lipid, R2 is
[0358] In some embodiments of the ionizable lipid, R2a is hydrogen.
[0359] In some embodiments of the ionizable lipid, R2a is methyl.
[0360] In some embodiments of the ionizable lipid, R2a is ethyl.
[0361] In some embodiments of the ionizable lipid, R2a is propyl.
[0362] In some embodiments of the ionizable lipid, R2a is butyl.
[0363] In some embodiments of the ionizable lipid, R2a is pentyl.
[0364] In some embodiments of the ionizable lipid, R2a is hexyl.
[0365] In some embodiments of the ionizable lipid, R2a is heptyl.
[0366] In some embodiments of the ionizable lipid, R2a is octyl.
[0367] In some embodiments of the ionizable lipid, R2b is hydrogen.
[0368] In some embodiments of the ionizable lipid, R2b is methyl.
[0369] In some embodiments of the ionizable lipid, R2b is ethyl.
[0370] In some embodiments of the ionizable lipid, R2b is propyl.
[0371] In some embodiments of the ionizable lipid, R2b is butyl.
[0372] In some embodiments of the ionizable lipid, R2b is pentyl.
[0373] In some embodiments of the ionizable lipid, R2b is hexyl.
[0374] In some embodiments of the ionizable lipid, R2b is heptyl.
[0375] In some embodiments of the ionizable lipid, R2b is octyl.
[0376] In some embodiments of the ionizable lipid, R2a is hydrogen and R2b is hydrogen,
[0377] In some embodiments of the ionizable lipid, R2a is hexyl and R2b is hydrogen.
[0378] In some embodiments of the ionizable lipid, R2a is octyl and R2b is hydrogen.
[0379] In some embodiments of the ionizable lipid, R2a is hydrogen and R2b is butyl.
[0380] In some embodiments of the ionizable lipid, R2c is methyl.
[0381] In some embodiments of the ionizable lipid, R2c is ethyl.
[0382] In some embodiments of the ionizable lipid, R2c is propyl.
[0383] In some embodiments of the ionizable lipid, R2c is butyl.
[0384] In some embodiments of the ionizable lipid, R2c is pentyl.
[0385] In some embodiments of the ionizable lipid, R2c is hexyl.
[0386] In some embodiments of the ionizable lipid, R2c is heptyl.
[0387] In some embodiments of the ionizable lipid, R2c is octyl. [0388] In some embodiments of the ionizable lipid, R2 is --( C1-6 alkylene)-(C3-8 cycloalkyl)- C1-6 alkyl.
[0389] In some embodiments of the ionizable lipid, R2 is --( C1-6 alkylene )-(cyclohexyl)-C1-6 alkyl.
[0390] In some embodiments of the ionizable lipid, R2 is -(C1-6 alkylene)-(cyclopentyl)-C1-6 alkyl.
Variables R3, R3a, R3b, and R3c
[0391] In some embodiments of the ionizable lipid, R3 is
[0392] In some embodiments of the ionizable lipid, R3a is hydrogen.
[0393] In some embodiments of the ionizable lipid, R3a is methyl.
[0394] In some embodiments of the ionizable lipid, R3a is ethyl.
[0395] In some embodiments of the ionizable lipid, R3a is propyl.
[0396] In some embodiments of the ionizable lipid, R3a is butyl.
[0397] In some embodiments of the ionizable lipid, R3a is pentyl.
[0398] In some embodiments of the ionizable lipid, R3a is hexyl.
[0399] In some embodiments of the ionizable lipid, R3a is heptyl.
[0400] In some embodiments of the ionizable lipid, R3a is octyl.
[0401] In some embodiments of the ionizable lipid, R3b is hydrogen.
[0402] In some embodiments of the ionizable lipid, R3b is methyl.
[0403] In some embodiments of the ionizable lipid, R3b is ethyl.
[0404] In some embodiments of the ionizable lipid, R3b is propyl.
[0405] In some embodiments of the ionizable lipid, R3b is butyl.
[0406] In some embodiments of the ionizable lipid, R3b is pentyl.
[0407] In some embodiments of the ionizable lipid, R3b is hexyl.
[0408] In some embodiments of the ionizable lipid, R3b is heptyl.
[0409] In some embodiments of the ionizable lipid, R3b is octyl.
[0410] In some embodiments of the ionizable lipid, R3a is octyl and R3b is hydrogen.
[0411] In some embodiments of the ionizable lipid, R3a is ethyl and R3b is hydrogen.
[0412] In some embodiments of the ionizable lipid, R3a is hexyl and R3b is hydrogen.
[0413] In some embodiments of the ionizable lipid, R3c is methyl.
[0414] In some embodiments of the ionizable lipid, R3c is ethyl. [0415] In some embodiments of the ionizable lipid, R3c is propyl.
[0416] In some embodiments of the ionizable lipid, R3c is butyl.
[0417] In some embodiments of the ionizable lipid, R3c is pentyl.
[0418] In some embodiments of the ionizable lipid, R3c is hexyl.
[0419] In some embodiments of the ionizable lipid, R3c is heptyl.
[0420] In some embodiments of the ionizable lipid, R3c is octyl.
[0421] It is understood that., for an ionizable lipid, variables o, R1, RN, RN , RN , m, n, M, M’, R2, R2a, R2b, R2C, R ’, R3a, R3b, and R3c can each be, where applicable, selected from the groups described herein, and any group described herein for any of variables O..R1, RN, RN , RN\ m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b. »3c can be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN’, RN , m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3c.
[0422] In some embodiments, the ionizable lipid is a compound selected from Table IL-1.
Table IL-1: Ionizable lipids
63
[0423] In some embodiments, the ionizable lipid is a compound selected from Table IL-2.
Table IL-2: Ionizable lipids
[0424] In some embodiments, the ionizable lipid is a compound of Formula (IL-IIA): (IL-IIA), or its N-oxide, or a salt or isomer thereof, wherein: m is selected from 5, 6, 7, 8, and 9;
R2 and R3 are each independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl;
R4 is selected from -(CH2)nOH, wherein n is selected from 1, 2, 3, 4, and 5, and wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R10 is -
N(R)2, wherein each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H;
M is selected from -OC(O)O-, -C(O)O~, -O-M”-O-, and -N(RM)C(O)-, in which M” is -(CH2)ZC(O)-, wherein z is 1, 2, 3, or 4;
M’ is selected from -OC(O)O-, -C(O)O-, -O-M”-O-, -N(RM)C(O)O-, and -O- N= C( R ” )-, wherein:
M” is -(CH2)zC(O>, C1-13 alkyl, -B(R**), or -Si(R**)2-; z is 1, 2, 3, or 4, each RMis independently selected from H and C1-6 alkyl; each R** is independently selected from H and C1-12 alkyl;
R’a is C1- 18 alkyl, C2-18 alkenyl, or -R*YR*”, wherein: each R*” is independently C1 -15 alkyl; each R* is independently C1-12 alkyl; each Y is independently a C3-6 carbocycle; and
R” is a C3-C13 alkyl, optionally substituted with OH.
[0425] In some embodiments, the ionizable lipid is a. compound of Formula (IL-IIAX): (IL-IIAX) or its N-oxide, or a salt or isomer thereof, wherein:
R1 is -R”M’R’, wherein: each R’ is independently C1 -18 alkyl;
M’ is selected from -C(O)O- and -0-N=:C(RM)-, wherein each RM is independently selected from H and C1-6 alkyl; each R” is independently C3-15 alkyl,
R2 and R3 are each independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl;
R4 is selected from -(CH2)nOH, wherein n is selected from 1, 2, 3, 4, and 5, and , wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and. R10 is - N(R)2, wherein each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; each R5 is H; each R6 is H; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.
[0426] In some embodiments, the ionizable lipid is a compound selected from Table IL-3.
Table IL-3: Ionizable lipids
[0427] In some embodiments, the ionizable lipid is a compound of Formula (IL-IIB): (IL-IIB) or its N-oxide, or a salt or isomer thereof, wherein R’a is: and R ’b is: wherein denotes a point of attachment;
Raβ, Ra γ, and Raδ are each independently selected from the group consisting of H,
C1-12 alkyl, and C2-12 alkenyl;
Rbβ, Rbγ and Rbδ are each independently selected from the group consisting of H,
C1-12 alkyl, and C2-12 alkenyl, wherein at least one of Rbβ, Rbγ and Rbδ is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
R2 and RJ are each independently selected from the group consisting of C1 -14 alkyl and
C2-14 alkenyl;
R4 is selected from -(CH2)nNRTQ, -(CH2)nNRS(O)2TQ, -(CH2)nNRC(O)H and
-(CH2)nNRC(O)TQ wherein n is selected from 1, 2, 3, 4, and 5;
T is a bond or a C1 -3 alkyl linker, C2-3 alkenyl linker, or C2-3 alkynyl linker; Q is selected from 3-14 membered heterocycle containing 1-5 heteroatoms selected from N, O, and S, C3-10 carbocycle, C1-6 alkyl, and C2-6 alkenyl, wherein the alkyl, alkenyl, heterocycle, and carbocycle are each optionally substituted with one or more RQ; each RQ independently is selected from the group consisting of oxo, hydroxyl, cyano, amino, C1-6 alkylamino, di-C1-6 alkylamino, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C1-6 alkanolyl, -C(O)C1-6 alkyl, and -NRC(O) C1-6 alkyl; each R is independently selected from H, C1-6 alkyl, and C2-6 alkenyl; each R’ is independently selected from C1-12 alkyl and C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9; and
1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.
[0428] In some embodiments, the ionizable lipid is a compound selected from Table IL-4.
Table IL-4: Ionizable lipids
[0429] In some embodiments, the ionizable lipid is a compound selected from Table IL-5.
Table IL-5: Ionizable lipids
[0430] In some embodiments, the ionizable lipid is a. compound of Formula (IL-IIC):
(IL-IIC) or its N-oxide, or a salt or isomer thereof. wherein: is , wherein denotes a point of attachment; wherein Raa and Rap are each independently selected from the group consisting of H and C1-2 alkyl, wherein at least one of Raa and R*3 is a C1 or C2 alkyl;
R’ is selected from the group consisting of C1 -18 alkyl and C2-18 alkenyl;
R2 and R3 are each independently selected from the group consisting of C1 -14 alkyl and C2-14 alkenyl;
R4 is -(CH2)nQ, wherein n is independently selected from 1, 2, 3, 4, and. 5, where Q is selected from NRS(O)2Rsx and , wherein A is a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S; and a is 1, 2, 3, or
4, wherein denotes a point of attachment;
R is selected from H and C1 -3 alkyl;
RsX is selected from a C3-8 carbocycle, a 3-14 membered, heterocycle containing one or more heteroatoms selected from N, O and S, C1-6 alkyl, C2-6 alkenyl, (C1-3 alkoxy)C1-3 alkyl, and wherein the carbocycle and heterocycle are optionally substituted with one or more groups selected from oxo, C1-6 alkyl, and (C1-3 alkoxy)C1-3 alkyl;
RSX 1 is selected from C(O)NR14R14’, a C3-8 carbocycle, and a 3-14 membered heterocycle containing one or more heteroatoms selected from N, O and S, wherein the carbocycle and heterocycle are each optionally substituted with one or more groups selected from oxo, halo, C1-3 alkyl, (C1-3 alkoxy )C1-3 alkyl, C1-6 alkylamino, di-(C1-6 alkyl) amino, and NH2; each Rk’ is selected from the group consisting of OH, oxo, halo, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C1-6 alkylamino, di-(C1-6 alkyl) amino, NH2, C(O)NH2, CM, and NO2;
R14 and R14 are each independently selected from the group consisting of H and C1-6 alkyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;
1 is selected, from 1, 2, 3, 4, 5, 6, 7, 8, and 9; p1 is selected from 1, 2, 3, 4, and 5; and
P2 is selected from 1, 2, 3, 4, and 5.
[0431] In some embodiments, the ionizable lipid is a. compound selected from Table IL-6.
Table IL-6: Ionizable lipids
[0432] In some embodiments, the ionizable lipid is a compound of Formula (IL-III): (IL-III), or salts or isomers thereof, wherein,
W is ring A is t is 1 or 2;
A1 and A2 are each independently selected, from CH or N;
Z is CH2 or absent wherein when Z is CH2, the dashed lines (1 ) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
R1, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R” MR', -R*YR”, -YR”, and -R*OR”;
Rx1 and Rx2 are each independently H or C1-3 alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, - OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, - P(O)(OR’)O-, -S(O)2-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;
M* is C1-C6 alkyl,
W1 and W2 are each independently selected from the group consisting of -O- and - N(R6)-; each R6 is independently selected from the group consisting of H and C1-5 alkyl;
X1, X2, and X3 are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O~, -OC(O)-, -(CH2)n-C(O)-, -C(O)-(CH2)n-, -(CH2)tl- C(O)O-, -OC(O)-(CH2)n-, -(CH2)n-OC(O)-, -C(O)O-(CH2)n-, -CH(OH)-, -C(S)-, and - CH(SH)-; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl and a C3-6 carbocycle; each R’ is independently selected from the group consisting of C1-12 alkyl, C2-12 alkenyl, and H; each R” is independently selected from the group consisting of C3-12 alkyl, C3-12 alkenyl and -R*M R‘. and n is an integer from 1-6.
[0433] In some embodiments, the ionizable lipid is a compound of Formula (IL-IIIA): or a. salt or isomer thereof, wherein R1, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR", -YR”, and -R*OR”; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O~, -C(O)N(R’)~, -N(R’)C(O)-, -C(O>, -C(S)-, -C(S)S-, -SC(S)
-CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an and group, and a heteroaryl group;
X1, X2, and X3 are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-. -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, -CH2-OC(O)-, -CH(OH)-, -C(S)~, and -CH(SH)-; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl and a C3-5 carbocycle; each R’ is independently selected from the group consisting of C1-12 alkyl, C2-12 alkenyl, and H; and each R” is independently selected from the group consisting of C3-12 alkyl and C3-12 alkenyl.
[0434] In some embodiments, the ionizable lipid is a compound selected from Table IL-7.
Table IL-7: Ionizable lipids
[0435] In some embodiments, the ionizable lipid is a compound selected from:
[0436] In some embodiments, the ionizable lipid is
[0437] In some embodiments, the ionizable lipid is
[0438] In some embodiments, the ionizable lipid is
[0439] In some embodiments, the ionizable lipid is
[0440] In some embodiments, the ionizable lipid is
[0441] Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and. a negative charge.
Polyethylene Glycol (PEG) Lipids
[0442] As used herein, the term “PEG lipid” refers 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. Such lipids are also referred to as PEGylated lipids. For example, a PEG- lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, or a PEG-DSPE lipid.
[0443] In some embodiments, the PEG lipid includes, but not limited to, 1,2-dimyristoyl-sn- glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxipropyl-3-amine (PEG-c-DMA) .
[0444] In some embodiments, the PEG lipid is selected from the group consisting of a PEG- modified phosphatidylethanol amine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof
[0445] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about. C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example, an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG lipid is PEG2k-DMG.
[0446] In some embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.
[0447] PEG lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.
[0448] In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described in International Patent Application No. PCT/US2016/000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,’ which is incorporated by reference in its entirety .
[0449] The lipid, component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG- lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanol amines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified di acyl glycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEGDMPE, PEG-DPPC, or a PEG-DSPE lipid.
[0450] In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:
[0451] In some embodiments, the PEG-modified lipids are a modified form of PEG-DSG.
PEG-DSG has the following structure:
[0452J In some embodiments, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG- chain. In some embodiments, the PEG lipid is a. PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy -PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (- OH) groups on the lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an OH group at. the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.
[0453] In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-I). Provided herein are compounds of Formula (PL-I): or salts thereof, wherein:
R3 is ORO
RO is hydrogen, optionally substituted alkyl, or an oxygen-protecting group; r is an integer between 1 and 100, inclusive;
L1 is optionally substituted C1 -10 alkylene, wherein at least one methylene of the optionally substituted C1 -10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene,
D is absent; or D is a moiety obtained by dick chemistry or a moiety cleavable under physiological conditions; m is O, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10;
A is of the formula: each instance of L2 is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with each instance of R2 is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2 are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen-protecting group;
Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.
[0454] In some embodiments, the compound of Formula (PL-I) is a PEG-OH lipid (i.e., R3 is -ORO, and RO is hydrogen). In some embodiments, the compound of Formula (PL-I) is of Formula (PL-I-OH):
(PL-I-OH), or a salt thereof.
[0455] In some embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In some embodiments, a PEG lipid useful in the present invention is a compound of Formula (PL-11). Provided herein are compounds of Formula (PL-II): (PL-II), or a salt thereof, wherein:
R3 is--ORO; RO is hydrogen, optionally substituted alkyl or an oxygen -protecting group, r is an integer between 1 and 100, inclusive;
R5 is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R' are replaced with optionally substituted, carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, , and each instance of RN is independently hydrogen, optionally substituted alkyl, or a nitrogen-protecting group .
[0456] In some embodiments, the compound of Formula (PL-II) is of Formula (PL-II-OH): (PL-II-OH), or a salt thereof. In some embodiments, r is 35-55. In some embodiments, r is 45.
[0457] In yet other embodiments the compound of Formula. (PL-II) is: (PL-01), or a salt thereof. In some embodiments, r is 1 -100. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45.
[0458] In some embodiments, the compound of Formula (PL-II) is
(PL-02).
[0459] In yet other embodiments, the PEG lipid is PEG1. PEG1 is a plurality of compounds of Formula (PL-01): or salts thereof, wherein r is 1-100. In some embodiments, r is about 35 to about 55. In some embodiments, r is 35-55. In some embodiments, r is 45.
[0460] In some embodiments, the PEG lipids may be one or more of the PEG lipids described, in U.S. Application No. 62/520,530.
Structural Lipids
[0461] As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties.
[0462] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.
[0463] In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 62/520,530.
Polynucleotides and Nucleic Acids
[0464] The lipid nanoparticles and pharmaceutical compositions comprising same of the disclosure can be used to deliver therapeutic and/or prophylactic agents to a target cell, tissue, organ or subject. The therapeutic agent can be a polynucleotide, such as mRNA or DNA. The polynucleotide can encode a polypeptide, such as a therapeutic agent or therapeutic polypeptide, or a CRISPR protein for use in gene editing.
[0465] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to human CD33+ hematopoietic stem cells.
[0466] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid to CD169+ marginal Macrophages. [0467] In some embodiments, LNPs comprising Compound No. 9 are suitable for delivery of nucleic acids encoding polypeptides to promote an immune response (e.g., any antigen described herein). In some embodiments, the LNPs improve the quality of the immune response.
[0468] In some embodiments, LNPs comprising Compound No. 1 are for delivery of nucleic acids encoding polypeptides for therapeutic use when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 1 show reduced immune response compared to LNPs that do not comprise Compound No. 1.
[0469] In some embodiments, LNPs comprising Compound No. 9 are for delivery of nucleic acids encoding polypeptides for therapeutic use when a dampened immune response is desired. In some embodiments, LNPs comprising Compound No. 9 show reduced immune response compared to LNPs that do not comprise Compound No. 9.
[0470] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
[0471] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells.
[0472] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a. nucleic acid encoding a. therapeutic polypeptide to hematopoietic stem and progenitor cells, e.g. HCD45+, Lineage negative, CD34+ and/or CD38- HSPCs.
[0473] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of a nucleic acid encoding a therapeutic polypeptide to erythroid progenitor cells.
[0474] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to myeloid progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38+ myeloid progenitor cells.
[0475] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a. CRISPR protein and/or gRNA to erythroid progenitor cells.
[0476] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acid encoding CRISPR protein and/or gRNA to long-term repopulating hematopoietic stem cells, e.g. hCD45+, Lineage negative, CD34+, CD38-, and/or CD45RA- long-term repopulating hematopoietic stem cells. [0477] In some embodiments, LNPs comprising Compound No. 1 are suitable for delivery of one or more nucleic acids encoding a CRISPR protein and/or gRNA to hematopoietic stem and. progenitor cells, e.g. hCD45+, Lineage negative, CD34+ and/or CD38-HSPCs.
[0478] In some embodiments, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates or produces de novo) protein expression. Non-limiting examples of types of therapeutic agents that can be used for enhancing protein expression include RNAs, mRNAs, dsRNAs, CRISPR/Cas based systems, as well as single and double stranded DNAs (e.g., expression vectors).
[0479] In some embodiments, therapeutic agent comprises an mRNA. The agent that upregulates protein expression may upregulate expression of a naturally occurring or non- naturally occurring protein (e.g., a chimeric protein that has been modified to improve half- life, a fusion protein, or one that comprises desirable amino acid changes). Exemplary proteins include intracellular, transmembrane, or secreted proteins, gene editing proteins, peptides, or polypeptides. The mRNA may encode the protein whose expression is upregulated, and upon delivery to the target cell, tissue, organ or subject, be translated to produced increased expression of the protein. Alternatively, the mRNA. may encode a protein that indirectly upregulates the expression of one or more proteins. For example, the mRNA may encode a natural or synthetic transcriptional activator.
[0480] In some embodiments, the therapeutic agent comprises a DNA therapeutic agent (e.g., a DNA molecule). The DNA molecule can be a double-stranded DNA, a single-stranded DNA. (ssDNA), or a molecule that is a partially double-stranded DNA, i.e., has a portion that is double-stranded and a portion that is single-stranded. In some cases, the DNA molecule is triple-stranded or is partially triple-stranded, i.e., has a portion that is triple stranded and a portion that is double stranded. The DNA molecule can be a circular DNA molecule or a linear DNA molecule.
[0481] A DNA therapeutic and/or prophylactic agent can be a DNA molecule that is capable of transferring a. gene into a cell, e.g., that encodes and can express a. transcript. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non- limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.
[0482] The DNA therapeutic and/or prophylactic agents described herein, e.g., DNA vectors, can include a variety of different features. The DNA therapeutic and/or prophylactic agents described herein, e.g., DNA vectors, can include a non-coding DNA sequence. For example, a DNA sequence can include at least one regulatory element for a. gene, e.g., a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, 5’ untranslated region (UTR), 3’ UTR and the like. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence comprises a pair of inverted terminal repeats (ITRs), e.g. of a transposon. In some embodiments, a DNA sequence described herein can have a non-coding DNA sequence that is operatively linked to a gene that is transcriptionally active. In other embodiments, a DNA sequence described herein can have a non-coding DNA sequence that is not linked to a gene, i.e., the non-coding DNA does not regulate a gene on the DNA. sequence.
[0483] In some embodiments, the one or more therapeutic and/or prophylactic agents are selected from the group consisting of a plasmid expression vector, a viral expression vector, and mixtures thereof
[0484] For example, in some embodiments, when the therapeutic and/or prophylactic agents comprise an RNA, the RNA is selected from the group consisting of a single-stranded RNA, a double-stranded RNA (dsRNA), a partially double-stranded RNA, and mixtures thereof. In some embodiments, the RNA is selected from the group consisting of a circular RNA, a linear RNA, and mixtures thereof.
[0485] For example, in some embodiments, when the therapeutic and/or prophylactic agents comprise an RNA, the RNA is selected from the group consisting of a spliceosomal RNA, a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), an RNA. interference (RNAi) molecule, a microRNA (miRNA ), an antagomir, an antisense RNA, a ribozyme, a Dicer substrate RNA (dsRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), locked nucleic acids (LNAs) and a guide RNA for a CRISPR/Cas system, and mixtures thereof.
[0486] For example, in some embodiments, when the therapeutic and/or prophylactic agents comprise an RNA, the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA. (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and mixtures thereof.
[0487] In some embodiments, the therapeutic and/or prophylactic agents comprise RNA and DNA, e.g. as an RNA-DNA hybrid.
[0488] In some embodiments, the one or more therapeutic and/or prophylactic agents comprise an mRNA. In some embodiments, the one or more therapeutic and/or prophylactic agents comprise a modified mRNA (mmRN.A). In some embodiments, the mRNA comprises Nl- methyl-pseudouridine. In some embodiments, the mRNA comprises nucleosides selected from the group consisting of N1-methyl-pseudouridine, adenosine, guanosine, and cytidine, for example in the open reading frame of the mRNA.
[0489] In some embodiments, the one or more therapeutic and/or prophylactic agents comprise an mRNA that incorporates a micro-RNA binding site (miR binding site). Further, in some embodiments, an mRNA includes one or more of a stem loop, a chain-terminating nucleoside, a poly A. sequence, a poly adenylation signal, and/or a 5' cap structure.
[0490] An mRNA may be a naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nudeobases, nucleosides, or nucleotides, as described below, in which case it may be referred to as a “modified mRNA” or “mmRNA.” As described herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g, a pentose or ribose) or derivative thereof in combination with an organic base (e.g, a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a. phosphate group.
[0491] An mRNA may include a 5' untranslated region (5'-UTR), a 3' untranslated region (5'- UTR), and/or a coding region (e.g., an open reading frame). An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000) of base pairs. Any number (e.g., all, some, or none) of nudeobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In some embodiments, all of a particular nucleobase type may be modified. In some embodiments, all uracils or uridines are modified. When all nudeobases, nucleosides, or nucleotides are modified, e.g, all uracils or uridines, the mRNA can be referred to as “fully modified,” e.g, for uracil or uridine.
[0492] In some embodiments, an mRNA, as described herein, may include a 5' cap structure, a chain-terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and/or a polyadenylation signal.
[0493] A 5' cap structure or cap species is a compound including two nucleoside moi eties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). A cap species may include one or more modified nucleosides and/or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes
[0494] An mRNA may instead or additionally include a chain-terminating nucleoside. For example, a chain-terminating nucleoside may include those nucleosides deoxygenated at the 2' and/or 3' positions of their sugar group. Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxy cytosine, 3' deoxyguanosine, 3' deoxythymine, and 2 ',3' dideoxynucleosides, such aass 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2',3' dideoxyguanosine, and 2',3' dideoxythymine. In some embodiments, incorporation of a chain-terminating nucleotide into an mRNA, for example at the 3 '-terminus, may result in stabilization of the mRNA.
[0495] An mRNA may instead or additionally include a stem loop, such as a histone 3’ UTR stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a. stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' untranslated region or a 3' untranslated region), a coding region, or a poly A sequence or tail. In some embodiments, a stem loop may affect one or more function(s) of an mRNA, such as nucleocytoplasmic transport, stability, initiation of translation, translation efficiency, and/or transcriptional termination.
[0496] An mRNA may instead or additionally include a poly A sequence and/or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A poly A sequence may also comprise stabilizing nucleotides or analogs. For example, a. poly A sequence can include deoxythymidine, e.g, inverted (or reverse linkage) deoxythymidine (d.T), as a stabilizing nucleotide or analog. Details on using inverted dT and other stabilizing poly A sequence modifications can be found, for example, in WO2017/049275 A2, the content of which is incorporated herein by reference. A polyA sequence may be a. tail located adjacent to a 3' untranslated region of an mRNA.. In some embodiments, a polyA sequence may affect the nuclear export, translation, and/or stability of an mRNA. Suitable polyA sequences will be known to persons of ordinary skill in the art and include, without limitation the bovine growth, hormone polyadenylation (bgh-PolyA) sequence, the SV40 polyadenylation sequence and the rabbit beta globin (rbGlob) polyadenylation sequence. [0497] An mRNA may comprise one or more post-transcriptional regulatory elements, including, without limitation, a nuclear export element such as Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or constitutive transport, element (CTE).
[0498] An mRNA may instead or additionally include a microRNA-binding site. MicroRNA- binding sites (or miR-binding sites) can be used to regulate mRNA expression in various tissues or cell types. In exemplary embodiments, miR-binding sites are engineered into 3’ UTR sequences of an mRNA to regulate, e.g., enhance degradation of mRNA in cells or tissues expressing the cognate miR. Such regulation is useful to regulate or control “off-target” expression in mRNAs, Le., expression in undesired cells or tissues in vivo. Details on using miR-binding sites can be found, for example, in WO 2017/062513 A2, the content of which is incorporated herein by reference.
[0499] In some embodiments, an mRNA is a bicistronic mRNA comprising a first coding region and a second coding region with an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows for internal translation initiation between the first and second coding regions, or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide (e.g. P2A., E2A, F2A and the like). IRES sequences and 2A. peptides are typically used express multiple proteins from the same vector. A variety of IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.
[0500] In some embodiments, an mRNA of the disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides (sometimes termed herein “modified mRNAs” or “mmRNAs”). In some embodiments, modified mRNAs may have useful properties, including enhanced stability, intracellular retention, enhanced translation, and/or the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, as compared to a reference unmodified mRNA. Therefore, use of modified mRNAs may enhance the efficiency of protein production, intracellular retention of nucleic acids, as well as possess reduced immunogenicity.
[0501] In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3 or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may have reduced degradation in a cell into which the modified mRNA is introduced, relative to a corresponding unmodified. mRNA. [0502] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ ), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g, 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethyl - pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm 5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio- uridine (cmnm5s2U), 5 -propynyl -uridine, 1 -propynyl -pseudouridine, 5-taurinomethyl-uridine (τm5U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1- taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1 -methyl -pseudouridine (mly/), 5-methyl-2-thio-uridine (m5s2U), 1-methyl- 4-thio-pseudouridi ne (m 1 s4y), 4-thio- 1 -methyl -pseudouridi ne, 3 -methyl-pseudouridine (m3\;/), 2-thi o- 1 -methyl-pseudouridine, 1 -methyl-1-deaza-pseudouridine, 2 -thio-1-methyl-1- deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5- methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxy -uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1 -methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1 - methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5-
(isopentenylaminomethyl)uridine (inmSU), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- m ethyl -pseudouridine 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-
2,-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncmSUm), 5- carboxymethylaminomethyl-2'-O-methyl -uridine (cmnmSUm), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.
[0503] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary' nucleobases and nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl -cytidine (ac4C), 5-formyl- cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5- iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1 -methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-1 -methyl-1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy -cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy -pseudoisocytidine, 4-methoxy-1-methyl- pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl- cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.
[0504] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a. modified adenine include a-thio-adenosine, 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g, 6-chloro-purine), 2-amino~6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl- adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl~adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6.A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynonvalylcarbamoyl- adenosine (hn6A), 2-methylthio-N6-hydroxynorvaly1carbamoyl-adenosine (ms2hn6A), N6- acetyl -adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a- thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2’-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (mlAm), 2'-O- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.
[0505] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include a-thio-guanosine, inosine (I), 1-methyl-inosine (m11), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (oQ), galactosyl -queuosine (galQ), mannosyl -queuosine (tnanQ), 7-cyano-7- deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (mlG), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl- guanosine (m2,2,7G), 8-oxo-guanosine, 7-methy1-8-oxo-guanosine, 1-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl- 2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (mlGm), N2,7-dimethyl- 2'-O-methyl-guanosine (m2,7Gm), 2,-O-methyl-inosine (Im), 1,2'-O-dimethyl -inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Grip)) , 1 -thio-guanosine, O6-methyl -guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.
[0506] In some embodiments, a modified mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (c.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
[0507] In some embodiments, the modified nucleobase is pseudouridine ( ψ), N1- methylpseudouridine (m1ψ ), 2-thiouridine, 4’ -thiouridine, 5-methylcytosine, 2-thio-1-methyl - 1 -deaza- pseudouridine, 2-thio-l -methyl -pseudouridine, 2-thio-5-aza-uridine , 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methyl -pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyl uridine. In some embodiments, an mRNA. of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases). In some embodiments, the modified nucleobase is N1- methylpseudouridine (m1ψ ) and the mRNA of the disclosure is fully modified with N1- methylpseudouridine (m1ψ ). In some embodiments, N1 -methylpseudouridine (m1ψ ) represents from 75-100% of the uracils in the mRNA. In some embodiments, N1- methylpseudouridine (m1ψ ) represents 100% of the uracils in the mRNA.
[0508] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2 -thio-cytidine (s2C), and 2-thio-5-methyl-cytidine. In some embodiments, a modified mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
[0509] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1 -methyl- adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (c.g. , a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
[0510] In some embodiments, the modified nucleobase is a modified, guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methyl -inosine (mil), wyosine (imG), methylwyosine (mim G), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1 ), 7-methyl-guanosine (m7G), 1 -methyl -guanosine (mlG), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
[0511] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (mli|/), 5- methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), a-thio-guanosine, or a-thio-adenosine. In some embodiments, an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g, a combination of 2, 3 or 4 of the aforementioned modified nucleobases).
[0512] In some embodiments, the modified mRNA comprises pseudouridine ( ψ). In some embodiments, the modified mRNA comprises pseudouridine (y) and 5-methyl-cytidine (rn5C). In some embodiments, the modified mRNA comprises 1-methyl-pseudouridine (m1ψ ). In some embodiments, the modified mRNA comprises 1-methyl-pseudouridine (m1ψ ) and 5-methyl- cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the modified mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the modified mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the modified mRNA comprises 2'-O-methyl uridine. In some embodiments, the modified mRNA comprises 2'-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the modified mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the modified mRNA comprises N6-methyl-adenosine (m6.A) and 5- methyl-cytidine (m5C).
[0513] In some embodiments, a modified mRNA of the disclosure is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification. For example, a. modified mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ ) or 5-methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with Nl-methylpseudouridine (m1ψ ) or 5-methyl-cytidine (m5C). Similarly, modified mRNAs of the disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
[0514] In some embodiments, a modified mRNA of the disclosure may be modified in a coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, a modified mRNA may be modified in regions besides a coding region. For example, in some embodiments, a 5'-UTR and/or a 3 -UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.
[0515] The modified mRNAs of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.
[0516] Where a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of that A, U , G or C nucleotide or nucleoside having been modified. Where percentages are listed, these represent the percentage of that particular A, U, G or C nucleobase triphosphate of the total amount of A, U, G, or C triphosphate present. For example, the combination: 25 % 5-Aminoallyl-CTP + 75 % CTP/ 25 % 5-Methoxy-UTP + 75 % UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of the uracils are UTP. Where no modified UTP is listed, then the naturally occurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of those nucleotides found in the polynucleotide. In this example, all of the GTP and ATP nucleotides are left unmodified.
[0517] The mRNAs of the present disclosure, or regions thereof, may be codon optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove proteins trafficking sequences, remove/add post translation modification sites in encoded proteins (e.g., glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome-binding sites and mRNA degradation sites, adjust translation rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and sendees are known in the art; non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA) and/or proprietary methods. In some embodiments, the mRNA sequence is optimized using optimization algorithms, e.g., to optimize expression in mammalian cells or enhance mRNA stability.
[0518] In some embodiments, the present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.
[0519] mRNAs of the present disclosure may be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In some embodiments, mRNAs are made using IVT enzymatic synthesis methods. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.
[0520] Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis. In some embodiments, modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugar. In particular embodiments, the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain, with chemical synthesis or with a polymerase enzyme.
[0521] Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Therapeutic -Agents for Reducing Protein Expression
In some embodiments, the therapeutic agent is a therapeutic agent that reduces (i.e., decreases, inhibits, or downregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used for reducing protein expression include mRNAs that incorporate a micro-RNA binding site(s) (miR binding site), microRNAs (miRNAs), antagomirs, small (short) interfering RNAs (siRNAs) (including shortmers and dicer-substrate RNAs), R'NA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs) and CRISPR/Cas9 technology.
Sensor Sequences and MicroRNA (miRNA) Binding Sites
[0522] In some embodiments, the prophylactic and/or therapeutic agent comprises a polynucleotide that binds, or encodes a protein that binds, to a target (sensor) sequence present in a target cell, organ, tissue or subject.
[0523] Sensor sequences include, for example, microRNA (miRNA.) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof. Non-limiting examples of sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
[0524] In some embodiments, a polyribonucleotide (e.g, a ribonucleic acid. (RNA), e.g, a messenger RNA (mRNA)) of the di sclosure comprising an open reading frame (ORF) encoding a polypeptide further comprises a sensor sequence. In some embodiments, the sensor sequence comprises a. miRNA-binding site.
[0525] A miRNA is a 19-25 nucleotide long noncoding RNA that binds to a polyribonucleotide and down -regulates gene expression, either by reducing stability or by inhibiting translation of the polyribonucleotide. A miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA. A miRNA. seed can comprise positions 2-8 or 2- 7 of the mature miRNA. In some embodiments, a miRNA seed can comprise 7 nucleotides (e.g, nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA-binding site is flanked by an adenosine (A) opposed to miRNA position 1. In some embodiments, a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA- binding site is flanked by an adenosine (A) opposed to miRNA position 1. See, for example, Crimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6;27(l):91-105. miRNA profiling of the target cells or tissues can be conducted to determine the presence or absence of miRNA. in the cells or tissues. In some embodiments, a polyribonucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprises one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences can correspond to any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of each of which are incorporated herein by reference in their entirety. [0526] As used herein, the term “microRNA (miRNA or miR) binding site” refers to a sequence within a polyribonucleotide, e.g, within a DNA or within an RNA transcript, including in the 5'UTR and/or 3'UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA. In some embodiments, a polyribonucleotide of the disclosure comprising an ORF encoding a. polypeptide further comprises a miRNA-binding site. In exemplary' embodiments, a 5'UTR and/or 3'UTR of the polyribonucleotide (e.g, a ribonucleic acid (RNA), e.g., a. messenger RNA (mRNA)) comprises a miRNA-binding site.
[0527] A miRNA-binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polyribonucleotide, e.g., miRNA-mediated translational repression or degradation of the polyribonucleotide. In exemplary embodiments of the disclosure, a miRNA-binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polyribonucleotide, e.g;, miRNA-guided RNA- induced silencing complex (RlSC)-mediated cleavage of mRNA. The miRNA-binding site can have complementarity to, for example, a. 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence. A miRNA-binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally occurring miRNA sequence. In some embodiments, the desired regulation is mRNA degradation. In some embodiments, the miRNA-binding site has full or complete complementarity (e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally occurring miRNA). In some embodiments, the mRNA degradation has full or complete complementarity.
[0528] In some embodiments, a miRNA-binding site includes a sequence that has complementarity (e.g, partial or complete complementarity) with a miRNA seed sequence. In some embodiments, the miRNA-binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a. miRNA-binding site includes a sequence that has complementarity partial or complete complementarity) with a miRNA sequence. In some embodiments, the miRNA-binding site includes a sequence that, has complete complementarity with a miRNA sequence. In some embodiments, a miRNA- binding site has complete complementarity with a miRNA sequence, but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
[0529] In some embodiments, the miRNA-binding site is the same length as the corresponding miRNA. In some embodiments, the miRNA-binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA-binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3’ terminus, or both. The miRNA-binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA-binding sites or preventing the mRNA from translation.
[0530] In some embodiments, the miRNA-binding site binds to the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA-binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA-binding site or prevents the mRNA from being translated. In some embodiments, the miRNA-binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polyribonucleotide comprising the miRNA-binding site. In some embodiments, the miRNA-binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polyribonucleotide comprising the miRNA-binding site. In another embodiment, the miRNA-binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polyribonucleotide comprising the miRNA-binding site.
[0531] In some embodiments, the miRNA-binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
[0532] In some embodiments, the miRNA-binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about, seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
[0533] By engineering one or more miRNA-binding sites into a polyribonucleotide of the disclosure, the polyribonucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polyribonucleotide. In some embodiments, if a polyribonucleotide of the disclosure is not intended to be delivered to a tissue or cell but ends up there, then a. miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5'UTR and/or 3' TR of the polyribonucleotide. [0534] Conversely, miRNA-binding sites can be removed from polyribonucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues. In some embodiments, a binding site for a specific miRNA can be removed from a polyribonucleotide to improve protein expression in tissues or cells containing the miRNA.
[0535] In one embodiment, a polyribonucleotide of the disclosure can include at least one miRNA-binding site in the 5'UTR and/or 3'UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells. In another embodiment, a polyribonucleotide of the disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5'~UTR and/or 3'-UTR in order to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
[0536] Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA-binding sites. The decision of whether to remove or insert a miRNA-binding site can be made based on miRNA expression patterns and/or their profilings in diseases. Identification of miRNAs, miRNA-binding sites, and their expression patterns and role in biology have been reported (c.g., Bonauer et al., Curr Drug Targets 2010 11 :943-949, .Anand and Cheresh Curr Opin Hematol 2011 18: 171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129: 1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393- 403 and all references therein; each of which is incorporated herein by reference in its entirety). [0537] miRNAs and miRNA-binding sites can correspond to any known sequence, including non-limiting examples described in U.S. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.
[0538] Examples of tissues where miRNA are known to regulate mRNA, and thereby protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR- 208), endothelial cells (miR- 17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR- 16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR- 1d, miR- 149), kidney (miR- 192, miR- 194, miR-204), and lung epithelial cells (let-7, miR-133, miR- 126).
[0539] Specifically, miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g, dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell-specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells-specific miRNAs also regulate many embodiments of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells). In some embodiments, miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polyribonucleotide can be shut off by adding miR-142-binding sites to the 3'-UTR of the polyribonucleotide, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polyribonucleotides in antigen-presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161, Brown BD, et al., Natmed. 2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
[0540] An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen-presenting cells and displayed on the surface of the antigen-presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
[0541] Introducing a miR-142-binding site into the 5'UTR and/or 3'UTR of a polyribonucleotide of the disclosure can selectively repress gene expression in antigen- presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen-presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polyribonucleotide. The polyribonucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.
[0542] In one embodiment, binding sites for miRNAs that are known to be expressed, in immune cells, in particular, antigen-presenting cells, can be engineered into a polyribonucleotide of the disclosure to suppress the expression of the polyribonucleotide in antigen-presenting cells through miRNA-mediated RNA degradation, subduing the antigen- mediated immune response. Expression of the polyribonucleotide is maintained in non- immune cells where the immune cell-specific miRNAs are not expressed. In some embodiments, to prevent an immunogenic reaction against a liver-specific protein, any miR- 122-binding site can be removed and a. miR-142 (and/or mirR-146)-binding site can be engineered into the 5'UTR and/or 3'UTR of a polyribonucleotide of the disclosure.
[0543] To further drive the selective degradation and suppression in APCs and macrophage, a polyribonucleotide of the disclosure can include a further negative regulatory; element in the 5'UTR and/or 3'UTR, either alone or in combination with miR-142 and/or miR-146 binding sites. As a non-limiting example, the further negative regulatory element is a Constitutive Decay Element (CDE).
[0544] Immune cell-specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let- 7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-Iet-7e-5p, hsa-let-7g-3p, hsa-Iet-7g-5p, hsa-let- 7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-1et-7f-l-- 3p, hsa-let-7f-2--5p, hsa-let-7f-5p, miR-125b-l-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR- 130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a- 5p, miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a- 3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-l-3p, miR-16-2-3p, miR-16-5p, miR-17- 5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p, miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p, miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-l-5p, miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p, miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a- 5p, miR-27b-3p, miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-l -5p, miR-29b-2-5p, miR-29c~3p, miR-29c-5p, miR-30e-3p, miR-30e-5p, miR-331- 5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346, miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:el 18-el27; Vaz C et al., BMC Genomics, 2010, 1 1 ,288, the content of each of which is incorporated herein by reference in its entirety).
[0545] miRNAs that are known to be expressed in the liver include, but are not limited to, miR- 107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR- 1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR- 199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p. miRNA-binding sites from any liver-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the liver. Liver-specific miRNA-binding sites can be engineered, alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure. [0546] miRNAs that are known to be expressed in the lung include, but are not limited to, let- 7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a- 3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p. MiRNA- binding sites from any lung-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the lung. Lung-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure. [0547] miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a- 5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. MiRNA-binding sites from any heart-specific microRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the heart. Heart-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g, APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
[0548] miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR- 125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR- 212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d- 5p, miR-329, miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383, miR- 410, miR-425~3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571, miR-7-l-3p, miR-7-2-3p, miR-7-5p, miR-802, miR- 922, miR-9-3p, and miR-9-5p. MiRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR- 148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR- 219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. MiRNA-binding sites from any CNS-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the nervous system. Nervous system-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
[0549] miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a- 5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. MiRNA- binding sites from any pancreas-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the pancreas. Pancreas-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
[0550] miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2- 3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR- 562, MiRNA-binding sites from any kidney-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the kidney. Kidney-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
[0551] miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR- 143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. MiRNA-binding sites from any muscle-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the muscle. Muscle-specific miRNA-binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA-binding sites in a polyribonucleotide of the disclosure.
[0552] miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes. [0553] miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17- 3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-115p, miR-19b-2-5p, miR- 19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p, miR-513a-5p, miR-92a-l-5p, miR-92a-2- 5p, miR-92a-3p, miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered in endothelial cells from deep sequencing analysis (e.g., Voellenkle C et. al., RNA, 2012, 18, 472- 484, herein incorporated by reference in its entirety). MiRNA-binding sites from any endothelial cell-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the endothelial cells.
[0554] miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR~34c~5p, miR~449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let- 7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. MiRNA-binding sites from any epithelial cell-specific miRNA can be introduced to or removed from a polyribonucleotide of the disclosure to regulate expression of the polyribonucleotide in the epithelial cells.
[0555] In addition, a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development, and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436; Goff LA et al., PLoS One, 2009, 4:e7192; Morin RD et al.. Genome Res, 2008, 18, 610-621; Yoo JK et al., Stem Cells Dev. 2012, 21(11), 2049- 2057, each of which is herein incorporated by reference in its entirety). MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let.7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR- 1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR- 200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367- 3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p, miR- 423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR- 548g-5p, miR-548i, miR-548k, miR-5481, miR-548m, miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766- 5p, miR-885-3p, miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p, miR- 99b-3p, and miR-99b-5p. Many predicted novel miRNAs are discovered by deep sequencing in human embryonic stem cells (e.g, Morin RD et al., Genome Res, 2008, 18, 610-621; Goff LA et al., PLoS One, 2009, 4:e7192; Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each of which is incorporated herein by reference in its entirety).
[0556] In one embodiment, the binding sites of embryonic stem cell-specific miRNAs can be included in or removed from the 3'UTR of a polyribonucleotide of the disclosure to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a. degenerative condition (e.g, degenerative diseases), or to stimulate the senescence and. apoptosis of stem cells in a disease condition (e.g., cancer stem cells).
[0557] Many miRNA expression studies are conducted to profile the differential expression of miRNAs in various cancer cells/tissues and other diseases. Some miRNAs are abnormally over-expressed in certain cancer cells and others are under-expressed. In some embodiments, miRNAs are differentially expressed in cancer cells (W02008/154098, US2013/0059015, US2013/0042333, WO201 1/157294), cancer stern cells (US2012/0053224); pancreatic cancers and diseases (US2009/0131348, 1182011/0171646, US2010/0286232, US8389210); asthma, and inflammation (US8415096); prostate cancer (US2013/0053264); hepatocellular carcinoma (WO2012/151212, US2012/0329672, W02008/054828, US8252538); lung cancer cells (WO2011/076143, WO2013/033640, W02009/070653, US2010/0323357); cutaneous T cell lymphoma (W02013/011378); colorectal cancer cells (WO2011/0281756, WO2011/076142); cancer positive lymph nodes (W02009/100430, US2009/0263803); nasopharyngeal carcinoma (EP2112235); chronic obstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroid cancer (WO2013/066678); ovarian cancer cells ( US2012/0309645, WO2011/095623); breast cancer cells (W02008/154098, W02007/081740, US2012/0214699), leukemia aanndd lymphoma (W02008/073915, US2009/0092974, US2012/0316081, US2012/0283310, WO2010/018563, the content of each of which is incorporated herein by reference in its entirety).
[0558] As a non-limiting example, miRNA-binding sites for miRNAs that are over-expressed in certain cancer and/or tumor cells can be removed from the 3'UTR of a polyribonucleotide of the disclosure, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death. Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected.
[0559] MiRNA can also regulate complex biological processes such as angiogenesis (e.g, miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176). In the polynucleotides or polyribonucleotides of the disclosure, miRNA-binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polyribonucleotides (or polyribonucleotides encoded by polynucleotides) to biologically relevant cell types or relevant biological processes. In this context, the polyribonucleotides of the disclosure are defined as auxotrophic polyribonucleotides.
Peptide/Polypeptide Therapeutic Agents
[0560] In some embodiments, the therapeutic and/or prophylactic agent is a peptide therapeutic agent. In some embodiments, the therapeutic and/or prophylactic agent is a polypeptide therapeutic agent. In some embodiments, the therapeutic agent is a protein. In some embodiments, the therapeutic agent can be delivered to (administered to) a subject, organ, tissue or cell using the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure as an encoding polynucleotide that is translated (and optionally transcribed if DNA) into the therapeutic polypeptide after delivery.
[0561] In some embodiments, the peptide or polypeptide is naturally derived, e.g, isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g. , a. synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g, contains less than 3, less than 5, less than 10, less than 15, less than 20, or less than 25 amino substitutions, deletions, or additions compared to its wild type, naturally occurring peptide or polypeptide counterpart).
[0562] In some embodiments, the protein comprises a wild type, or substantially wiki type protein, or a protein which retains substantially wild type activity, that is administered to a subject with a disease characterized by a deficiency in the expression or activity of the protein. In some embodiments, the disease is a rare disease, i.e. a disease that effects a small proportion of the population. In many cases, rare diseases are caused by mutations in autosomal recessive genes that, lead to loss of gene activity , and can thus be treated by supplying the missing gene activity. For example, the lipid nanoparticles, pharmaceutical compositions and. methods of the disclosure can be used to supply the protein product from the mutated gene, or a DNA or mRNA encoding the protein.
[0563] In some embodiments, the disease compri ses Propionic acidemia (PA), Methylmalonic acidemia (MMA), Glycogen Storage Disease Type la (GSDla), Ornithine transcarbamylase deficiency (OTC), Phenylketonuria (PKU), or Crigler-Najjar Syndrome Type 1 (CN-1).
[0564] In some embodiments, the disease comprises Propionic acidemia (PA), and the protein comprises rpropionyl-CoA carboxylase subunit alpha. (PCC A) and/or propionyl-CoA carboxylase subunit beta (PCCB), or a variant or derivative thereof.
[0565] In some embodiments, the disease comprises Methylmalonic acidemia (MMA), and the protein comprises methylmalonyl-CoA mutase (MMUT
[0566] In some embodiments, the disease comprises Glycogen Storage Disease Type la (GSDla), and the protein comprises enzyme glucose-6-phosphatase (G6Pase), or a. derivative or variant thereof.
[0567] In some embodiments, the disease comprises Ornithine transcarbamylase deficiency, and the protein comprises ornithine transcarbamylase (OTC), or a variant or derivative thereof. [0568] In some embodiments, the disease comprises Phenylketonuria. (PKU), and the protein comprises phenylalanine hydroxylase (PAH), or a variant or derivative thereof.
[0569] In some embodiments, the disease comprises Crigler-Najjar Syndrome Type 1 (CN-I), and the protein comprises UDP glucuronosyltransferase family 1 member Al (UGT1A1), or a variant or derivative thereof.
[0570] In some embodiments the polypeptide comprises a fusion protein. Exemplary fusion proteins include, but are not limited, to, fusion proteins comprising DNA binding domains (e.g., TALEN, or transcription activator-like effector nuclease DNA. binding domains. Zinc Finger Nuclease, or ZFP DNA binding domains, or catalytically inactive Class 2 CRISPR/Cas systems) fused to one or more effector domains, such as transcriptional activation, repression or base editing domains, and the like. Further examples of fusion proteins include proteins with fusion domains to enhance in vivo stability and/or bioavailable of the protein (e.g., Fc fusion proteins), and fusion proteins with enhanced tissue, organ or cellular specificity (e.g., antibody fusion proteins). Genome Editing Techniques
[0571] The lipid nanoparticles, pharmaceutical compositions and methods of the disclosure can be used to deliver (administer) gene editing system to a subject organ, tissue or cell, thereby editing the genome of the target cell, or cells of the subject, organ or tissue.
[0572] Suitable gene editing systems will be known to persons of ordinary skill in the art, and include, but are not limited to, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems.
[0573] As used herein, “clustered regularly interspaced short palindromic repeats /CRISPR associated” or “CRISPR/Cas system” and similar terms refer to genome editing systems derived from adaptive immune systems present in bacteria and archaea. A typical CRISPR/Cas system has two components: an effector protein (or protein complex, depending on the system) which can cleave a target nucleic acid sequence, and a. guide nucleic acid (or nucleic acids), usually RNA, which contain a targeting sequence complementary' to the target sequence and which bind the effector protein(s) and guide the effector protein(s) to the corresponding target. The guide nucleic acid (gNA) can be one nucleic acid (a single gNA) or multiple nucleic acids (e.g., a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA)) depending on the CRISPR/Cas system. A gNA typically includes a scaffold sequence, which interacts with the CRISPR protein, and an approximately 20 nucleotide targeting sequence that is complementary to, and capable of hybridizing to, the target sequence. [0574] CRISPR/Cas systems can be classified into 2 classes (Class 1 and Class 2), 6 types (I to VI) and several subtypes, with multi-Cas protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI) (see, for example, Li et al. Comput Struct Biotechnol J. 2020 Sep 8,18:2401 -2415, the contents of which are incorporated by reference herein in their entirety). Optionally, the nuclease activity of the CRISPR/Cas system can be modified or eliminated to produce a CRISPR/Cas system capable of binding, but not cleaving, or nicking rather than cleaving, a target nucleic acid. Such catalytically inactive CRISPR/Cas systems have utility, for example as DNA binding domains incorporated into fusion proteins. Exemplary Class 2 systems suitable for use with the lipid nanoparticles and pharmaceutical compositions of the disclosure include, but are not limited to Cas9 (including SpCas9, SaCas9, FnCas9 and NmCas9), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (CasMini), Casl3a (C2c2), Cast 3b (C2c4), Cas13d and their derivatives. Additional CRISPR/Cas systems derived from bacteriophages (e.g., CasPhi) are also contemplated as within the scope of the instant disclosure. Selection of suitable guide nucleic acid sequences (e.g., gRNA) and targeting sequences based on the proto-spacer adjacent motif (PAM) specificity of the Cas protein are within the skill of persons of ordinary skill in the art.
[0575] In some embodiments, the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure deliver (administer) a nucleic acid to a subject, organ, tissue or cell that, is part of a gene editing system, such as the CRISPR/Cas systems described herein. In some embodiments, the nucleic acid comprises at least one nucleic acid suitable for a genome editing technique selected from the group consisting of an mRNA. encoding a gene editing protein (e.g. a ZFN, TALEN or CRISPR protein), a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a single guide RNA (sgRNA), and a DNA repair template, or a combination thereof. In some embodiments, the nucleic acid comprises an mRNA encoding a protein of a gene editing system, such as a CRISPR protein. In some embodiments, the nucleic acid comprises DNA encoding the gene editing protein, such as a. CRISPR protein. In some embodiments, the nucleic acid comprises a gNA (or a component of gNA, where the gNA is not a. single gNA).
[0576] In some embodiments, the gene editing comprises use of a DNA repair pathway. DNA repair pathways, which occur after double-strand breaks in the target DNA molecule, include, but are not limited to, homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In some embodiments, the DNA repair is comprises a template (e.g., HR). In such embodiments, the gene editing system comprises a DNA template molecule (e.g., one which includes a wild type version or desirable mutation, such as an insertion, deletion or base-pair substitution), of the target DNA to be edited. In some embodiments, the DNA template molecule is delivered to (administered to) the subject, organ, tissue or cell using the lipid nanoparticles, pharmaceutical compositions and. methods of the disclosure.
[0577] In some embodiments, the gene editing system comprises a modified gene editing system that is catalytically inactive, and can be used for epigenetic regulation, rather than editing of, a target nucleic acid in a. subject, organ, tissue or cell. For example, the CRISPR protein can be catalytically inactivated and fused to an effector domain, or the DNA binding domain of the ZFN or TALEN can be fused to a domain other than a nuclease domain. For example, the DNA binding domains of the gene editing systems described herein can be fused to one or more transcriptional activator domains (e.g., VP16) or repressor domains (KRAB, SETDB1, DNMT1, DMT3A and the like). Such systems are described, for example, in WO2022140577 and US20070192880, the contents of which are incorporated by reference in their entirety herein. [0578] In some embodiments, multiple components of the gene editing system (e.g., the CRISPR protein, or an mRNA or DNA encoding same, as well as the gNA, and optionally the DNA template molecule) are incorporated into the same sialic lipid nanoparticle. Alternatively, the components of the gene editing system can be incorporated into different sialic lipid nanoparticles.
Vaccines
[0579] In one embodiment, the disclosure provides a vaccine comprising a messenger ribonucleic acid (mRNA) formulated in a lipid nanoparticle or pharmaceutical composition of the disclosure. In some embodiments, the lipid nanoparticle comprises Compound 9. The mRNA can comprise an open reading frame encoding an antigen, for example a cancer antigen or an infectious disease antigen as described herein. Without wishing to be bound by theory it is thought that delivery of antigens to myeloid cells by the lipid nanoparticles, pharmaceutical compositions and methods of the disclosure can improve the quality of the immune response, e.g., by reducing Th2 cytokine production, while maintaining the desired level of Th l cytokine production and overall numbers of activated T cells.
[0580] Exemplary antigens are described in W02019036670, the contents of which are incorporated by reference in their entirety herein.
[0581] In some embodiments, the antigen comprises an infectious disease antigen, for example a virus or bacteria. In some embodiments, the infectious disease antigen comprises a protein, or a. portion of a protein, from the agent (bacteria, virus, parasitic eukaryote etc.) to which an immune response is desired.
[0582] In some embodiments, the infectious disease comprises an infectious disease that primarily affects adults, an infectious disease that primarily affects children and/or adolescents (adolescent and pediatric infectious disease), or an infectious disease that affects both adults and children. In some embodiments, the infectious diseases comprises a coronavirus (e.g., SARS-CoV-2 virus, which causes COVID-19), an influenza, virus (e.g., influenza. A, B, C or D), a respiratory syncytial virus (RSV), or a human metapneumovirus (hMPV). In some embodiments, the infectious disease comprises disease caused by a latent virus, i.e. a virus that is present in a resting state in the body. While such viruses may be present in a latent, i.e. no replicative state with no or minimal symptoms, they may reactivate upon external stimulus or when the immune sy stem is compromised. In some embodiments, the latent virus comprises a cytomegalovirus (CMV), an Epstein-Barr virus (EBV), a herpes simplex vims (HSV), e.g. HSV type 2, a varicella zoster vims (VZV), or a. human immunodeficiency vims (HIV) such as HIV-1 or HIV-2. In some embodiments, the infectious disease comprises an enteric virus, for example a norovirus. In some embodiments, the infectious disease comprises a bacteria. Exemplary bacteria, include, but are not limited to Borrelia burgdorferi, which cause lyme disease. In some embodiments, the disease is a disease that has caused, or has the potential to cause, a public health crisis due to the ease with which such viruses spread in the population and the severity of the illness such viruses cause. For example, viruses such as Zika virus, Nipah virus and monkeypox (Mpox) have all caused, or have the potential to cause public health problems.
[0583] In other embodiments, the virus is a strain of Influenza A or Influenza B or combinations thereof. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or fragment thereof. In some embodiments, the hemagglutinin protein is , or a portion thereof.
[0584] In some embodiments, the virus comprises coronavirus (e.g., SARS-CoV-2 virus and variants or strains thereof), and the antigen comprises the coronavirus spike protein (S) or a portion thereof. SARS-CoV-2 antigens are described, for example, in WO2022155524, the contents of which are incorporated by reference in their entirety.
[0585] In some embodiments, the virus comprises RSV or hMPV, and the antigen comprises a viral polyprotein or a portion thereof. Additional RSV and hMPV antigens are described in US20180326045, the contents of which are incorporated by reference in their entirety.
[0586] In some embodiments, the virus comprises CMV, and the antigen comprises one or more CMV glycoproteins.
[0587] In some embodiments, the antigen comprises cancer antigen. Examples of cancer antigens to which immune responses can be directed are known in the art., Sometimes referred to as tumor antigens, are molecules found in tumor cells that trigger an immune response in the subject with the tumor. Tumor antigens can be tumor associated antigens (TAAs), which are expressed in healthy tissues but overexpressed in cancer cells, or tumor specific antigens (TSA), which are only found in cancer cells.
[0588] In some embodiments, the cancer antigen (and resulting vaccine) is individualized, i.e. derived from and specific to the cancer of the subject. Individualized vaccines, for instance, may include RNA encoding for one or more known cancer antigens specific for the tumor or cancer antigens specific for each subject, referred to as neoepitopes or patient specific epitopes or antigens. A "patient specific cancer antigen" is an antigen that has been identified as being expressed in a. tumor of a particular patient. The patient specific cancer antigen may or may not be typically present in tumor samples generally. Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced in comparison to that in cancerous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes. Exemplary cancers that can be treated with individualized cancer vaccines include, but are not limited to, melanoma, nonsmall cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), renal cell carcinoma (R.CC), bladder cancer and solid tumors (early and late).
[0589] In some embodiments, the cancer antigen comprises a checkpoint antigen. Vaccines to checkpoint antigens can stimulate effector T cells that target and kill suppressive immune and cancer cells that express high levels of checkpoint. In some embodiments, the checkpoint antigen comprises IDO (indoleamine 2,3-dioxygenase 1) or a fragment thereof, PD-L1 (CD274 molecule), or a. fragment thereof, or combinations thereof.
[0590] In some embodiments the antigen or epitope is based on specific mutations (neoepitopes) and those expressed by cancer- germline genes (antigens common to tumors found in multiple patients).
[0591] An epitope, also known as an antigenic determinant, as used herein, is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells. Epitopes include B cell epitopes and T cell epitopes. B-cell epitopes are peptide sequences which are required for recognition by specific antibody producing B -cells. B cell epitopes refer to a specific region of the antigen that is recognized by an antibody. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody- are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous, i.e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.
[0592] T-cell epitopes are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I. The peptide epitope may be any length that is reasonable for an epitope. [0593] In some embodiments the peptide epitope is 9-30 amino acids. In other
[0594] embodiments the length is 9- 22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21, 10-20, 1 1 -22, 22-21, 1 1 -20, 12-22, 12-21, 12-20,13-22, 13-21, 13- 20, 14-19, 15-18, or 16-17 amino acids.
[0595] The of the disclosure may include mRNA sequences encoding one or more antigens. In some embodiments the mRNA comprises the sequence of 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more antigens. In other embodiments, the mRNA comprises sequences of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less cancer antigens. In yet other embodiments the mRNA comprises sequences of 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40- 100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15- 50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100- 500, 50-500, 50-800, 50-1,000, or 100-1,000 antigens.
Other Components
[0596] A lipid nanoparticle may include one or more components in addition to those described in the preceding sections. In some embodiments, a lipid nanoparticle may include one or more small hydrophobic molecules such as a vitamin (e.g, vitamin A or vitamin E) or a. sterol .
[0597] Lipid nanoparticles may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components. A permeability enhancer molecule may be a molecule described by U.S. patent application publication No. 2005/0222064, for example. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g, glycogen and derivatives and analogs thereof).
[0598] A polymer may be included in and/or used to encapsulate or partially encapsulate a lipid nanoparticle. A polymer may be biodegradable and/or biocompatible. A polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. In some embodiments, a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLG.A), poly(L -lactic acid-co-glycolic acid) (PLLGA), poly(D,L- lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L- lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-la.ctide), poly(D,L- lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PEL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes, such as polyethylene and polypropylene, polyalkylene glycols, such as polyethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates, such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters, such as poly(vinyl acetate), polyvinyl halides, such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), poly siloxanes, polystyrene, polyurethanes, derivatized celluloses, such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, pol oxamers, pol oxamines, poly(ortho)esters, poly(butyric acid), polyfvaleric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(/V-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol .
[0599] Surface altering agents may include, but are not limited to, anionic proteins (e.g. , bovine serum albumin), surfactants (e.g, cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g, cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rliDNase). A surface altering agent may be disposed within a nanoparticle and/or on the surface of a lipid nanoparticle (e.g., by coating, adsorption, covalent linkage, or other process).
[0600] A lipid nanoparticle may also comprise one or more functionalized lipids. In some embodiments, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a lipid, nanoparticle may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.
[0601] In addition to these components, lipid nanoparticles may include any substance useful in pharmaceutical compositions. In some embodiments, the lipid nanoparticle may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art. (See, for example, Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.)
[0602] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and/or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.
[0603] Surface active agents and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g, bentonite [aluminum silicate] and. VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g, stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, propylene glycol monostearate, and polyvinyl alcohol), carbomers (e.g, carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and methylcellulose), sorbitan fatty acid, esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, and sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g, CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRU® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof.
[0604] A binding agent may be starch (e.g., cornstarch and starch paste), gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, and mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti. gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol, inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.
[0605] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chiorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SEES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II NEOLONE™, KATHON™, and/or EUXYL®.
[0606] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and/or combinations thereof. Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.
[0607] Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea. tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimetbicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
Methods of Preparing the Lipid Nanopartides
[0608] In some embodiments, the present disclosure provides a. method of preparing the population of lipid nanoparticles described herein.
[0609] In some embodiments, the method comprises: i) mixing an ionizable lipid, a structural lipid, and a phospholipid, with a first buffer, thereby forming a population of intermediate empty lipid nanoparticles.
[0610] In some embodiments, the method comprises: i) mixing an ionizable lipid, a structural lipid, a phospholipid, and a PEG lipid, with a first buffer, thereby forming a population of intermediate empty lipid nanoparticles.
[0611] In some embodiments, the method further comprises: ii) adding a second buffer to the intermediate empty lipid nanoparticles, thereby forming a population of empty lipid nanoparticles.
[0612] In some embodiments, the method further comprises: iii) mixing a therapeutic agent (e.g., a nucleic acid) with the empty-lipid nanoparticles, thereby forming a population of loaded lipid nanoparticles.
[0613] In some embodiments, the method further comprises processing the empty lipid nanoparticles or the loaded lipid nanoparticles.
[0614] In some embodiments, the step of processing comprises: a) adding a cryoprotectant to the empty lipid nanoparticles or the loaded lipid nanopartides; b) lyophilizing the empty lipid nanoparticles or the loaded lipid nanoparticles; c) storing the lyophilized empty lipid nanoparticles or the lyophilized loaded lipid nanoparticles; and/or d) adding a buffering solution to the lyophilized empty lipid nanoparticles or the lyophilized loaded lipid nanoparticles.
[0615] Suitable methods for preparing the population of lipid nanoparticles described herein are also described in PCT Application Publication No. WO/2020/160397, WO/2021/155274, and WO/2022/032087, each of which is incorporated herein by reference.
Pharmaceutical Compositions
[0616] In some embodiments, the present disclosure provides a. pharmaceutical composition, comprising the population of lipid nanoparticles described herein, and one or more pharmaceutically acceptable carriers or excipients.
[0617] In some embodiments, the pharmaceutical composition is free of therapeutic agent (e.g, RNA).
[0618] In some embodiments, the pharmaceutical composition comprises a. therapeutic agent (e.g., RNA).
[0619] Pharmaceutical compositions may include one or more lipid nanoparticles. In some embodiments, a pharmaceutical composition may include one or more lipid nanoparticles including one or more different therapeutics and/or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21st Edition, A.. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a. lipid nanoparticle in the formulation of the disclosure. An excipient or accessory ingredient may be incompatible with a component of a. lipid nanoparticle of the formulation if its combination with the component or lipid nanoparticle may result in any undesirable biological effect or otherwise deleterious effect.
[0620] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid nanoparticle. In some embodiments, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved, for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United. States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0621] Relative amounts of the one or more lipid nanoparticles, the one or more pharmaceutically acceptable excipients, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition comprises between 0.1% and 100% (wt/wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition comprises between 0.1% and 15% (wt/vol) of one or more amphiphilic polymers (e.g, 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% w/v).
[0622] In some embodiments, the lipid nanoparticles and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g, being stored at a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30
°C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). For example, the pharmaceutical composition comprising one or more lipid, nanoparticles is a solution or solid (e.g, via lyophilization) that, is refrigerated for storage and/or shipment at, for example, about -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, or -80 °C. In some embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles and by storing the lipid nanoparticles and/or pharmaceutical compositions thereof at. a temperature of 4 °C or lower, such as a temperature between about -150 °C and about 0 °C or between about -80 °C and about -20 °C (e.g., about -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C
-70 °C, -80 °C, -90 °C, -130 °C or -150 °C).
[0623] Lipid nanoparticles and/or pharmaceutical compositions including one or more lipid nanoparticles may be administered, to any patient or subject including those patients or subjects that may benefit from a. therapeutic effect provided by the delivery of a therapeutic and/or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of lipid nanoparticles and pharmaceutical compositions including lipid nanoparticles are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary , if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and/or rats.
[0624] A pharmaceutical composition including one or more lipid nanoparticles may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and/or packaging the product into a desired single- or multi-dose unit.
[0625] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g, lipid nanoparticle). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.
[0626] Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. In some embodiments, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and. granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.
[0627] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms comprise inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and. sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include additional therapeutics and/or prophylactics, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In some embodiments for parenteral administration, compositions are mixed, with solubilizing agents, such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
[0628] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed, as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
[0629] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0630] In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and. crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drag in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug rel ease can be controlled. Examples of other biodegradabl e polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
[0631] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients, such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and. therefore melt in the rectum or vaginal cavity and release the active ingredient.
[0632] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least- one inert, pharmaceutically acceptable excipient, such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g, cetyl alcohol and glycerol monostearate), absorbents (e.g, kaolin and bentonite clay and silicates), and. lubricants (e.g, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.
[0633] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a. composition that they release the active ingredient(s) only. In some embodiments, the solid compositions may optionally comprise opacifying agents and. can be of a. composition that, they release the active ingredient(s) in a certain part, of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[0634] Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled, delivery of a compound, to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
[0635] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum comeum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144, 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941 ,880, 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. .Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.
[0636] Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations, such as liniments, lotions, oil in water and/or water in oil emulsions, such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (wt/wt) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
[0637] A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a. formulation may comprise dry particles which comprise the active ingredient. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self- propelling solvent/powder dispensing container, such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Dry' powder compositions may include a solid fine powder diluent, such as sugar, and. are conveniently provided in a. unit dose form. [0638] Low-boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally, the propellant may constitute 50% to 99.9% (wt/wt) of the composition, and active ingredient may constitute 0.1% to 20% (wt/wt) of the composition. A propellant may further comprise additional ingredients, such as a liquid non- ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
[0639] Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a. flavoring agent, such as saccharin sodium, a volatile oil, a buffering agent, a surface-active agent, and/or a preservative, such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 1 nm to about 200 nm.
[0640] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. Such a formulation is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
[0641] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (wt/wt) and as much as 100% (wt/wt) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may- be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may comprise, for example, 0.1% to 20% (wt/wt) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
[0642] A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0. 1/1.0% (wt/wt) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein. Other ophthalmically administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this present disclosure.
Methods of Producing Polypeptides in Cells
[0643] The present disclosure provides methods of producing a polypeptide of interest in a. mammalian cell. Methods of producing polypeptides involve contacting a cell with a formulation of the disclosure comprising a. lipid nanoparticle including an mRNA encoding the polypeptide of interest. Upon contacting the cell with the lipid nanoparticle, the mRNA may be taken up and translated in the cell to produce the polypeptide of interest. In some embodiments, the polypeptide of interest is a protein.
[0644] In general, the step of contacting a mammalian cell with a. lipid nanoparticle including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticle contacted with a cell, and/or the amount of mRNA. therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the lipid nanoparticle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors. In general, an effective amount of the lipid nanoparticle will allow for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.
[0645] The step of contacting a lipid nanoparticle including an mRNA. with a cell may involve or cause transfection. A phospholipid including in the lipid component of a lipid nanoparticle may facilitate transfection and/or increase transfection efficiency, for example, by interacting and/or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell.
[0646] In some embodiments, the lipid nanoparticles described herein may be used therapeutically or prophylactically. For example, an mRNA included in a lipid, nanoparticle may encode a therapeutic polypeptide (e.g, in a translatable region) and produce the therapeutic polypeptide upon contacting and/or entry (e.g., transfection) into a cell. In other embodiments, an mRNA included in a lipid nanoparticle may encode a polypeptide that may improve or increase the immunity of a. subject.
[0647] In some embodiments, contacting a cell with a lipid nanoparticle comprising a sialic acid lipid may effectuate a reduced inflammatory response, as measured by expression of Sca- 1, which is a surrogate marker of inflammatory reaction, as compared to the inflammatory' response effectuated by a lipid nanoparticle that does not comprise a sialic acid lipid.
Methods of Delivering Therapeutic Agents to Cells and Organs
[0648] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a cell in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
[0649] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to a cell in a subject.
[0650] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to a cell in a subject.
[0651] The LNPs of the present disclosure can be used to deliver nucleic acid payloads to cell populations in which expression or function of such nucleic acid molecules is desired. The subject LNPs represent an improvement over alternative delivery vehicles (e.g., LNPs of alternative composition) as they deliver payloads to desired cell lines and yet do not increase expression of markers known to be associated with inflammation, e.g., Sca-1.
[0652] In some embodiments, the LNPs of the present disclosure are used to deliver nucleic acid payload molecules to immune cells, e.g., to myeloid and lymphoid cells.
[0653] In some embodiments, payload molecules are delivered to hematopoietic stem and progenitor cell (HSPC) populations.
[0654] In some embodiments, the LNPs of the present disclosure can be used to deliver payload to the group(s) of cells which are referred to variously in the art and include: multipotent progenitors ; hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-; hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS); LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin- cKit+Scal-)); and.
Erythroid progenitor cells (EPCs).
[0655] In another embodiment, the subject LNPs are used to deliver mRNA payload to mature myeloid cells, e.g., monocytes and macrophages. In another embodiment, the subject LNPs are used to deliver mRNA payload to macrophages.
[0656] LNPs of the present disclosure may comprising a sialic acid lipid, an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid.
[0657] In some embodiments, LNPs comprising a sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to a multipotent progenitor cell.
[0658] In some embodiments, LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0659] In some embodiments, LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0660] In some embodiments, LNPs comprising a. sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin- cKit+Scal-)).
[0661] In some embodiments, LNPs comprising a sialic acid lipid (e.g., Compound 1), an ionizable lipid, a structural lipid, a PEG lipid, and a phospholipid are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0662] In some embodiments, the LNPs comprise a combination of ionizable lipid, PEG-lipid, sialic acid lipid, and phospholipid as specified in Table 1A”:
Table 1A”
[0663] Exemplary embodiments of LNPs of the present disclosure include those specified in
Table 1A:
Table 1A
[0664] In some embodiments, type A’ LNPs are used to deliver art mRNA payload to a multipotent progenitor cell.
[0665] In some embodiments, type A’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0666] In some embodiments, type A’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0667] In some embodiments, type A’ LNPs are used to deliver an mRNA payload to LK cells
( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0668] In some embodiments, type A.’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0669] In some embodiments, type B’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0670] In some embodiments, type B’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-. [0671] In some embodiments, type B’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and. multipotent progenitors, (also known as LKS).
[0672] In some embodiments, type B’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0673] In some embodiments, type B’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0674] In some embodiments, type E’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0675] In some embodiments, type E’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0676] In some embodiments, type E’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0677] In some embodiments, type E’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0678] In some embodiments, type E’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0679] In some embodiments, type F’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0680] In some embodiments, type F’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0681] In some embodiments, type F’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0682] In some embodiments, type F’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0683] In some embodiments, type F’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0684] In some embodiments, type G’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell. [0685] In some embodiments, type G’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0686] In some embodiments, type G’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0687] In some embodiments, type G’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0688] In some embodiments, type G’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0689] In some embodiments, type J’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0690] In some embodiments, type J’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0691] In some embodiments, type J’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0692] In some embodiments, type J’ LNPs are used to deliver an mRN A payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0693] In some embodiments, type J’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs).
[0694] In some embodiments, type K’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0695] In some embodiments, type K’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0696] In some embodiments, type K’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0697] In some embodiments, type K’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0698] In some embodiments, type K’ LNPs are used to deliver an mRNA payload to Erythroid progenitor cells (EPCs). [0699] In some embodiments, type L’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0700] In some embodiments, type L’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0701] In some embodiments, type L’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0702] In some embodiments, type L’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0703] In some embodiments, type L’ LNPs are used to deliver an mRNA payload to Erythroid, progenitor cells (EPCs).
[0704] In some embodiments, type M’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0705] In some embodiments, type M’ LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0706] In some embodiments, type M’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multipotent progenitors, (also known as LKS).
[0707] In some embodiments, type M’ LNPs are used to deliver an mRNA payload to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal-)).
[0708] In some embodiments, type M’ LNPs are used to deliver an mRNA payload to Erythroid, progenitor cells (EPCs).
[0709] In some embodiments, type N’ LNPs are used to deliver an mRNA payload to a multipotent progenitor cell.
[0710] In some embodiments, type N' LNPs are used to deliver an mRNA payload to hematopoietic stem cells (HSCs), also referred to herein as LT-HSCs (long term HSCs) having the phenotype Lin-cKit+Scal+CD150+CD48-.
[0711] In some embodiments, type N’ LNPs are used to deliver an mRNA payload to hematopoietic stem progenitor cells (HSPCs), which population contains HSCs and multi potent progenitors, (also known as LKS).
[0712] In some embodiments, type N’ LNPs are used to deliver an mRNA payload, to LK cells ( i.e., lineage (negative) c-kit (positive) myeloid progenitor cells (Lin-cKit+Scal -)). [0713] In some embodiments, type N’ L'NPs are used to deliver an mRNA payload to Eiythroid progenitor cells (EPCs).
[0714] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC) in a subject, comprising administering to the subject, the population of lipid nanoparticles or pharmaceutical composition described herein.
[0715] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to hematopoietic stem and progenitor cells (HSPC) in a subject.
[0716] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to hematopoietic stem and progenitor cells (HSPC) in a subject. [0717] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to an erythroid progenitor cell (EPC) in a. subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
[0718] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to an eiythroid progenitor cells (EPC) in a subject.
[0719] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to eiythroid progenitor cells (EPC) in a subject.
[0720] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to myeloid cells in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein.
[0721] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to myeloid cells in a subject.
[0722] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to myeloid cells in a subject.
[0723] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to lymphoid cells in a subject, comprising administering to the subject the population of lipid nanoparticles or pharmaceutical composition described herein. [0724] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described herein for use in delivering a therapeutic agent to lymphoid cells in a subject.
[0725] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or composition described herein in the manufacture of a medicament for delivering a therapeutic agent to lymphoid cells in a subject.
[0726] In some embodiments, the subject is human. The present disclosure provides methods of delivering a therapeutic and/or prophylactic, such as a nucleic acid, to a mammalian cell or organ. Delivery- of a therapeutic and/or prophylactic to a cell involves administering a formulation of the disclosure that comprises a lipid nanoparticle including the therapeutic and/or prophylactic, such as a nucleic acid, to a subject, where administration of the composition involves contacting the cell with the composition. In some embodiments nucleic acid, (such as an RNA, e.g., mRNA) may be delivered to a cell or organ. In the instance that a therapeutic and/or prophylactic is an mRNA, upon contacting a cell with the lipid nanoparticle, a translatable mRNA may be translated in the cell to produce a polypeptide of interest, e.g., a protein.
[0727] In some embodiments, a lipid nanoparticle may target a particular type or class of cells (e.g, cells of a particular organ or system thereof). In some embodiments, a lipid nanoparticle including a therapeutic and/or prophylactic of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, bone marrow, or lung. Specific delivery to a particular class of cells, an organ, or a. system or group thereof implies that a. higher proportion of lipid nanoparticles including a therapeutic and/or prophylactic are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of a lipid nanoparticle to a mammal. In some embodiments, specific delivery may result in a greater than 2 -fold, 5- fold, 10-fold, 15-fold, or 20-fold increase in the amount of therapeutic and/or prophylactic per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g, the spleen). In some embodiments, the tissue of interest is selected from the group consisting of a liver, kidney, bone marrow, a lung, a spleen, a femur, vascular endothelium in vessels (e.g, intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g, via intratumoral injection).
Methods of Treating Diseases and Disorders
[0728] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need. thereof the population of lipid nanoparticles or pharmaceutical composition described herein (e.g., in a therapeutically effective amount).
[0729] In some embodiments, the present disclosure provides the population of lipid nanoparticles or pharmaceutical composition described, herein for use in treating or preventing a disease or disorder in a subject.
[0730] In some embodiments, the present disclosure provides use of the population of lipid nanoparticles or pharmaceutical composition described herein in the manufacture of a medicament for treating or preventing a disease or disorder.
[0731] In some embodiments, the population of lipid nanoparticles or pharmaceutical composition is administered parenterally.
[0732] In some embodiments, the population of lipid nanoparticles or pharmaceutical composition is administered intramuscularly, intradermally, subcutaneously, and/or intravenously.
[0733] Lipid nanoparticles may be useful for treating a disease, disorder, or condition. In particular, such compositions may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity, a. disease that can be improved by gene editing or mRNA delivery to hematopoietic stem cells, or by administration of a. therapeutic or prophylactic vaccine.. In some embodiments, a. formulation of the disclosure that comprises a lipid nanoparticle including an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA. may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide.
[0734] The disclosure provides methods involving administering lipid nanoparticles including one or more therapeutic and/or prophylactic agents, such as a nucleic acid, and pharmaceutical compositions including the same. The terms therapeutic and prophylactic can be used interchangeably herein with respect to features and embodiments of the present disclosure. Therapeutic compositions, or imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any reasonable amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition and/or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of the administration; the particular composition; the mode of administration; and the like. Compositions in accordance with the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of a. composition of the present disclosure will be decided by an attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g, for imaging) for any particular patient will depend upon a variety of factors including the severity and identity of a disorder being treated, if any, the one or more therapeutics and/or prophylactics employed; the specific composition employed; the age, body weight, general health, sex, and diet of the patient, the time of administration, route of administration, and rate of excretion of the specific pharmaceutical composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific pharmaceutical composition employed, and like factors well known in the medical arts.
[0735] The disclosure provides methods involving administering lipid nanoparticles including the gene editing systems described herein, for example the CRISPR/Cas systems therein. The methods can be used to edit target nucleic acids. For example, the methods can be used to edit target nucleic acids such as nucleic acids comprising CD33 sequences in hematopoietic stem and progenitor cells (HSPC), erythroid progenitor cells (EPC), myeloid cells, and/or lymphoid cells and related cell types as described herein.
[0736] The disclosure provides methods involving administering lipid nanopartides including vaccines, as described herein. The lipid nanoparticles can comprise a mRNA comprising an open reading frame comprising a sequence encoding a cancer antigen or infectious disease antigen.
[0737] In some embodiments, compositions, including prophylactic, diagnostic, or imaging compositions including one or more lipid nanoparticles described herein, are administered by one or more of a variety of routes, including intravenous, intramuscular, or subcutaneous administration. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the lipid nanoparticle including one or more therapeutics and/or prophylactics (e.g, its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g, whether the patient, is able to tolerate particular routes of administration), etc.
[0738] Lipid nanoparticles including one or more therapeutics and/or prophylactics, such as a nucleic acid, may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at. the same time and/or formulated for delivery together, although these methods of delivery' are within the scope of the present disclosure. In some embodiments, one or more lipid nanoparticles including oonnee or more different, therapeutics and/or prophylactics may be administered in combination. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
[0739] It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.
[0740] The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that, the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating ccaanncceerr may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects, such as infusion related reactions).
[0741] A lipid nanoparticle may be used in combination with an agent to increase the effectiveness and/or therapeutic window of the composition. Such an agent may be, for example, an anti-inflammatory compound, a steroid (e.g., a corticosteroid), a statin, an estradiol, a BTK inhibitor, an SIP1 agonist, a glucocorticoid receptor modulator (GRM), or an anti-histamine, In some embodiments, a lipid nanoparticle may be used in combination with dexamethasone, methotrexate, acetaminophen, an HI receptor blocker, or an H2 receptor blocker. In some embodiments, a. method of treating a subject in need thereof or of delivering a therapeutic and/or prophylactic to a subject (e.g., a mammal) may involve pre-treating the subject with one or more agents prior to administering a lipid nanoparticle. In some embodiments, a subject may be pre-treated with a useful amount (e.g., 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other useful amount) of dexamethasone, methotrexate, acetaminophen, an Hl receptor blocker, or an H2 receptor blocker. Pre-treatment may occur 24 or fewer hours (e.g, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) before administration of the lipid nanoparticle and may occur one, two, or more times in, for example, increasing dosage amounts.
Biological Assays
[0742] Lipids, lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions described herein can be characterized using a variety of assays known to those skilled in the art to determine whether the compounds have biological activity. For example, the lipids, lipid nanoparticles, populations of lipid nanoparticles, and pharmaceutical compositions can be characterized by conventional assays, including but not limited to those assays described below', to determine whether they have a predicted activity, binding activity and/or binding specificity.
[0743] Furthermore, high-throughput screening can be used to speed up analysis using such assays. As a result, it can be possible to rapidly screen the molecules described herein for activity, using techniques known in the art. General methodologies for performing high- throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker; and U.S. Patent No. 5,763,263. High-throughput assays can use one or more different assay techniques including, but not limited to, those described below.
[0744] Various in vitro or in vivo biological assays may be suitable for detecting the effect of the compounds of the present disclosure. These in vitro or in vivo biological assays can include, but are not limited to, enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.
[0745] In some embodiments, the biological assay is described in the Examples herein.
[0746] In some embodiments, the biological assay is a transfection assay. In some embodiments, the biological assay is a. bone marrow transfection assay. In some embodiments, the assay is a spleen cell transfection assay. In some embodiments, the assay is an immune cell transfection assay.
[0747] In some embodiments, the biological assay is used to determine cell type, for example HSPC, LT-HSC, myeloid progenitor, erythroid progenitor or any other type of immune cell. In some embodiments, the biological assay comprises identifying expression of a plurality of markers by flow cytometry or immunofluorescence techniques. Suitable markers are known in the art, and described in the Examples. Definitions
[0748] A “vaccine” refers to a composition that is administered to a subject to stimulate the body’s immune response against a specific infectious agent or disease, thereby providing the subject with active acquired immunity to the infectious agent or disease.
[0749] “Reactogenicity” refers to the capacity of a vaccine to produce common, short-term adverse reactions shortly after administration, including, but not limited to, immunological responses such as fever, sore injection site, injection site bruising, redness, swelling, and the like.
[0750] “Immunogenicity” refers to the ability of a substance, such as an antigen delivered by vaccine, to cause the body to produce an immune response. With respect to vaccines, immunogenicity refers to the ability of an antigen to provoke an immune response in the subject upon administration of the vaccine. Immunogenicity can be influenced by multiple characteristics, including, but not limited to, molecule size, epitope density, chemical composition, antigen structure, and the presence of synthetic polymers. In the case of peptide antigens, the ability of the peptide to be proceed and presented to T cells via the major histocompatibility (MHC) complex can also play a role.
[0751] The term “vector” generally refers to a nucleic acid molecule capable of being transported between cells. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which virally-derived DNA or RNA sequences are present in the vector for packaging into a vims (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a. host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host, cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they contain or to which they are operatively linked. Such vectors are generally referred to as “expression vectors.” Common expression vectors include, but are not limited to, plasmids.
[0752] A “transposon” or “transposable element” refers to a DNA sequence that can change its position in a host genome. A typical wild-type transposon includes a transposase gene flanked by inverted terminal repeats (ITRs). The transposase recognizes the ITRs, and excises the transposon DNA body, which is then inserted at a new genomic location. When used to deliver a therapeutic agent to a target genome, the sequence encoding the transposase is typically replaced by the sequence of the therapeutic agent. Suitable transposons for delivery of therapeutic agents will be known to persons of ordinary skill in the art and include, but are not limited to, PiggyBac, Sleeping Beauty, Minos, Mariner and the like.
[0753] “RNAi” or “RNA interference” refers to the process of sequence-specific post- transcriptional gene silencing, mediated by double-stranded RNA (dsRNA). Duplex RNA siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA- directed RNA), pi RNA (Pi wi -interacting RNA), or rasiRNA (repeat associated siRNA) and modified forms thereof are all capable of mediating RNA interference. These dsRNA molecules may be commercially available or may be designed and prepared based on known sequence information, etc. The anti-sense strand of these molecules can include RNA, DNA, PNA, or a combination thereof. The RNA molecules can also include one or more modified nucleotides, as described herein, which can be incorporated on either strand.
[0754] In the RNAi gene silencing or knockdown process, dsRNA comprising a first (anti- sense) strand that is complementary to a portion of a. target gene and a second (sense) strand that is fully or partially complementary to the first anti-sense strand is introduced into a cell, tissue, organ or subject. After introduction, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed throughout the organism, and decrease mRNA of target gene, leading to a phenotype that may come to closely resemble the phenotype arising from a complete or partial deletion of the target gene.
[0755] Certain dsRNAs in cells can undergo the action of Dicer enzyme, a ribonuclease III enzyme. Dicer can process the dsRNA into shorter pieces of dsRNA, i.e. siRNAs. RNAi also involves an endonuclease complex known as the RNA. induced silencing complex (RISC). Following cleavage by Dicer, siRNAs enter the RISC complex and direct cleavage of a single stranded RNA target having a sequence complementary' to the anti-sense strand of the siRNA duplex. The other strand of the siRNA is the passenger strand. Cleavage of the target RNA takes place in the middle of the region complementary' to the anti-sense strand of the siRNA duplex. siRNAs can thus down regulate or knock down gene expression by mediating RNA interference in a sequence-specific manner.
[0756] As used herein, “complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A ” It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
[0757] As used herein, the terms “substantially complementaty” or “partially complementaty” mean that two nucleic acid sequences are complementary at least at about 50%, 60%, 70%, 80% or 90% of their nucleotides. Two nucleic acid sequences can be complementary at least at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides. For example, the two nucleic acid sequences can be between 60% to 100% complementary, between 70% to 100% complementary, between 80% and 100% complementary, between 90% and 100% complementary, between 60% to 90% complementary, between 60% to 80% complementary, between 60% and 70% complementary, between 70% and 90% complementary, between 70% and 80% complementaty, between 80% and 100% complementary , or between 80% and 90% complementary.
[0758] The terms “substantially complementary ” and “partially complementaty” can also mean that two nucleic acid sequences can hybridize under high stringency conditions, and such conditions are well known in the art.
[0759] As used herein, “heterologous” refers to a nucleic acid sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell. Thus, a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced, is heterologous with respect to that cell and the cell's descendants. In addition, a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, and/or under the control of different regulatory sequences than that found in nature.
[0760] As used herein, the term “lipid nanoparticle” or “LNP” refers to a nanoparticle comprising one or more lipids. In some embodiments, the LNP has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, or about 100 nm or less. In some embodiments, the LNP has a. size ranging from about 1 nm to about 100 nm.
[0761] As used herein, the term “liposome” refers to a composite having at least one lipid bilayer. In some embodiments, the liposome has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about. 200 nm or less, about 150 nm or less, or about 100 nm or less. In some embodiments, the liposome has a size ranging from about 1 nm to about 100 nm.
[0762] As used herein, the term “total lipids” refers to the collection of sialic acid lipids, ionizable lipids, structural lipids, and phospholipids, and PEG lipids in a given composition (e.g., a population of lipid nanoparticles).
[0763] As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1- 14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.
[0764] The term “heteroalkyd” refers to an alkyl group, as defined herein, wherein at least one carbon atom has been replaced by a heteroatom selected from the group consisting of oxygen, nitrogen, or sulfur. The nitrogen atom may be substituted or unsubstituted (e.g, NR wh erein R is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidised (i.e., N-->O and S(O)p, where p = 1 or 2). A divalent heteroalkyl is referred to herein as “heteroalkylene.”
[0765] As used herein, the term “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. The notation “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. In some embodiments, C18 alkenyl may include one or more double bonds. A C18 alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.
[0766] As used herein, the term “carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation “C3-6 carbocycle” means a. carbocycle including a. single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term “cycloalkyl” as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, Le., optionally substituted carbocycles.
[0767] As used herein, the term “heterocycle” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g, heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The term “heterocycloalkyl” as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.
[0768] As used herein, a “biodegradable group” is a group that, may facilitate faster metabolism of a lipid in a mammalian entity. .A biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, - C(S)-, -C(S)S-, -SC'(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group. As used herein, an “aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a “heteroaryl group” is an optionally substituted heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. In some embodiments, M and M’ can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M’ can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.
[0769] Alkyl, heteroalkyl, alkenyl, and cyclyl (e.g, carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of but are not. limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl, - OH), an ester (e.g, -C(O)OR -OC(O)R), an aldehyde (e.g.,-C(O)H), a carbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g., -C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., - OR), an acetal (e.g., -C(OR)2R””, in which each OR is alkoxy groups that can be the same or different and R”” is an alkyl or alkenyl group), a phosphate (e.g., P(O)43-), a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a. sulfinic acid (e.g, -S(O)OH), a sulfonic acid (e.g, -S(O)2OH), a thial (e.g., -C’(S)H), a sulfate (e.g., S(O)4 2-), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2 or -N(R)C(O)R), an azido (e.g, -Na), a nitro (e.g., -N02), a cyano (e.g., -CN), an isocyano (e.g, -NC), an acyl oxy (e.g.,-OC(O)R), an amino (e.g., -NR2, -NRH, or -NH2), a carbamoyl (e.g., - OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, - S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R. is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. In some embodiments, a C1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.
[0770] As used herein, the terms “approximately” and “about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In some embodiments, when used in the context of an amount of a given compound in a lipid component of a lipid nanop article, “about” may mean 10% of the recited value. For instance, a lipid nanoparticle including a. lipid component having about 40% of a given compound may include 30-50% of the compound.
[0771] As used herein, the term “compound” is meant to include all isomers and isotopes of the structure depicted. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. In some embodiments, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
[0772] As used herein, the term “upon” intends to refer to the time point being after an action happens. For example, “upon administration” refers to the time point being after the action of administration.
[0773] As used herein, the term “contacting” means establishing a physical connection between two or more entities. In some embodiments, contacting a mammalian cell with a lipid nanoparticle means that the mammalian cell and a nanoparticle are made to share a physical connection. Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts. In some embodiments, contacting a lipid nanoparticle and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g, intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of lipid nanoparticles. Moreover, more than one mammalian cell may be contacted by a lipid nanoparticle.
[0774] As used herein, the term “comparable method” refers to a method with comparable parameters or steps, as of the method being compared (e.g, producing the lipid nanoparticle formulation of the present disclosure). In some embodiments, the “comparable method” is a method with one or more of steps i), ia), iaa), ib), ii), iia), iib), lie), iid), and. iie) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps i), ia), iaa), ib), ii), iia), iib), iic), iid), and iie) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps ia) and ib) of the method being compared. In some embodiments, the “comparable method” is a method employing a. water-soluble salt of a nucleic acid. In some embodiments, the “comparable method” is a method employing an organic solution that does not comprise an organic solvent-soluble nucleic acid. In some embodiments, the “comparable method” is a method comprising processing the lipid nanoparticle prior to administering the lipid nanoparticle formulation. [0775] As used herein, the term “delivering” means providing an entity to a. destination. In some embodiments, delivering a therapeutic and/or prophylactic to a subject may involve administering a lipid nanoparticle including the therapeutic and/or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a lipid nanoparticle to a mammal or mammalian cell may involve contacting one or more cells with the lipid nanoparticle.
[0776] As used herein, “encapsulation efficiency" refers to the amount, of a therapeutic and/or prophylactic that becomes part of a nanoparticle composition, relative to the initial amount of therapeutic and/or prophylactic used in the preparation of a nanoparticle composition. For example, if 97 mg of therapeutic and/or prophylactic are encapsulated in a nanoparticle composition out of a 100 mg of therapeutic and/or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, "encapsulation" may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.
[0777] As used herein, "encapsulation," "encapsulated," "loaded," and "associated" may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement. As used herein, "encapsulation" or "association" may refer to the process of confining an individual nucleic acid molecule within a nanoparticle and/or establishing a physiochemical relationship between an individual nucleic acid molecule and a nanoparticle. As used herein, an "empty nanoparticle” may refer to a. nanoparticle that is substantially free of a therapeutic or prophylactic agent. As used herein, an "empty nanoparticle" or an "empty lipid nanoparticle" may refer to a nanoparticle that is substantially free of a nucleic acid. As used herein, an "empty nanoparticle" or an "empty lipid nanoparticle” may refer to a nanoparticle that is substantially free of a nucleotide or a polypeptide. As used herein, an "empty nanoparticle" or an "empty lipid nanoparticle" may refer to a nanoparticle that, consists substantially of only lipid components. -As used herein, a "loaded nanoparticle" or a "loaded lipid nanoparticle" (also referred to as a "full nanoparticle" or a “full lipid nanoparticle”) may refer to a. nanoparticle comprising the components of the empty nanoparticle, and a therapeutic or prophylactic agent. As used herein, a "loaded nanoparticle" or a. "loaded lipid nanoparticle" (also referred to as a "full nanoparticle" or a "full lipid nanoparticle") may refer to a nanoparticle comprising the components of the empty nanoparticle, and a nucleotide or polypeptide. As used herein, a "loaded nanoparticle" or a "loaded lipid nanoparticle" (also referred to as a. "full nanoparticle" or a "full lipid nanoparticle") may refer to a nanoparticle comprising the components of the empty nanoparticle, and a nucleic acid. [0778] As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide or protein and/or post-translational modification of a polypeptide or protein.
[0779] As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
[0780] As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
[0781] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.
[0782] As used herein, the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and/or double bonds and may thus exist as stereoisomers, such as double- bond isomers ( i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis! trans isomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known.
[0783] As used herein, a “lipid component” is that, component of a lipid nanoparticle that includes one or more lipids. In some embodiments, the lipid component may include one or more cationic/ionizable, PEGylated, structural, or other lipids, such as phospholipids.
[0784] “Lineage positive” or “lineage negative,” when used in reference to myeloid cells, refers to cellular expression of a plurality of markers used to assess the identity of cells derived from the major hematopoietic lineages. Exemplary cells that express the lineage markers include, without limitation, T cells, B cell, monocytes or macrophages, NK cells, erythrocytes and granulocytes. Stem cells from which the lineage positive cells differentiate (e.g., HSPCs, long-term repopulating hematopoietic stem cells, myeloid progenitor cells and the like) do not express, or minimally express, lineage markers. An exemplary set of lineage markers includes, but is not limited to CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56 and CD235a, or a combination or subset thereof. A further exemplary set of lineage markers includes CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD61, CD66b and. glycophorin A, or a. combination or subset thereof. Expression of these markers can be assessed by antibodies that bind the lineage antigens, followed by any suitable assay known in the art, such as immunofluorescence or flow cytometry.
[0785] As used herein, a ‘linker” is a moiety connecting two moi eties, for example, the connection between two nucleosides of a cap species. A linker may include one or more groups including but not limited to phosphate groups (e.g, phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphonates), alkyl groups, amidates, or glycerols. In some embodiments, two nucleosides of a cap analog may be linked at their 5’ positions by a triphosphate group or by a chain including two phosphate moieties and a boranophosphate moiety.
[0786] As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method, of administration may be selected to target delivery (e.g, to specifically deliver) to a. specific region or system of a body.
[0787] .As used herein, “modified” means non-natural. In some embodiments, an RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. A. “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. In some embodiments, a modified nucleobase species may include one or more substitutions that are not naturally occurring.
[0788] As used herein, the “N:P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g, in a. lipid nanoparticle including a lipid component and an RNA.
[0789] .As used herein, “naturally occurring” means existing in nature without artificial aid.
[0790] As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, or will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
[0791] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.
[0792] As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some embodiments, the polymeric lipid is a lipid comprising a polymer component. In some embodiments, the polymeric lipid is a PEG lipid. In some embodiments, the polymeric lipid is not a PEG lipid. In some embodiments, the polymeric lipid is Brij or OH- PEG-stearate. [0793] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, composition, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complication, commensurate with a reasonable benefit/risk ratio.
[0794] The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending, complexing, or dissolving the active compound) and having the properties of being substantially non toxic and non-inflammatory in a patient. Excipients may include, for example, anti -adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary’ excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.
[0795] Compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts. As used herein, “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, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, 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. Generally, 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. In some embodiments, the nonaqueous media are ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. 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. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
[0796] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid or an analog or derivative thereof may include choline. A phospholipid or an analog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane. In some embodiments, a cationic phospholipid may interact with one or more negatively charged phospholipids of a. membrane (e.g, a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.
[0797] As used herein, the "poly dispersity index," or "PDI," is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g, less than 0.3, indicates a narrow particle size distribution. [0798] As used herein, an amphiphilic “polymer” is an amphiphilic compound that comprises an oligomer or a polymer. In some embodiments, an amphiphilic polymer can comprise an oligomer fragment, such as two or more PEG monomer units. In some embodiments, an amphiphilic polymer described herein can be PS 20.
[0799] Unless indicated otherwise, and as one of ordinary skill in the art would understand, the number of repeating units indicated in the structure of a polymer refers to the average number of repeating units (a.k.a., average degree of polymerization). For example, a PEG lipid of the following structure refers to a plurality of polymers with an average chain length of 45 ethylene gly col units. E.g., in some embodiments, r is an integer from about 35 to about 55.
[0800] As used herein, the term “polypeptide” or “polypeptide of interest” refers to a. polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.
[0801] As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring. In some embodiments, 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 poly A sequence, and/or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. In some embodiments, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. 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, long non-coding RNA (IncRNA) and mixtures thereof
[0802] As used herein, the term “subject” refers to any organism to which a composition or formulation in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
[0803] As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human. In some embodiments, the organism is a patient.
[0804] As used herein, “target tissue” refers to any one or more tissue types of interest in which the delivery of a therapeutic and/or prophylactic would result in a desired biological and/or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In particular applications, a target tissue may be a kidney, a lung, a spleen, vascular endothelium in vessels (e.g., intra-coronary or intra-femoral), or tumor tissue via intratumoral injection). An “off-target tissue” refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and/or pharmacological effect. In particular applications, off-target tissues may include the liver and the spleen.
[0805] The term “therapeutic agent” or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents are also referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drags, proteins, and nucleic acids.
[0806] As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g, nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
[0807] As used herein, the term “transfection” refers to the introduction of a species (e.g., an RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
[0808] As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. In some embodiments, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated, with the disease, disorder, and/or condition.
[0809] As used herein, the term “zeta potential” refers to the surface charge of colloidal dispersions. The magnitude of the zeta, potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in the dispersion. Zeta potential can be measured on a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes the mobility and zeta potential by the principle of “Massively Parallel Phase Analysis Light Scattering” or MP-PALS. This measurement is more sensitive and less stress-inducing than ISO Method 13099-1 :2012 which only uses one angle of detection and required higher voltage for operation. In some embodiments, the zeta potential of the herein described empty lipid nanoparticle compositions lipid is measured using an instrument employing the principle of MP-PALS. Zeta potential can be measured on a Malvern Zetasizer (Nano ZS).
[0810] It is understood that some properties of lipid nanoparticles disclosed herein may be characterized by capillary zone electrophoresis (CZE). Capillary zone electrophoresis (CZE) refers to a separation technique which uses high voltage across a capillary to separate charged, species based on their electrophoretic mobility. In some embodiments, the CZE is conducted with an acetate buffer (e.g, 50mM sodium acetate at pH 5). In some embodiments, the CZE is conducted with a reverse voltage of about 10kV across a 75um capillary of 20cm effective length. In some embodiments, the capillaty is coated with polyethyleneimine.
[0811] The term “mobility peak,” as used herein, refers to a. peak representing the distribution of a substance (e.g, a population of lipid nanoparticles) as measured by CZE. In some embodiments, the intensity of the mobility peak is detected by scattered light. It is understood that the intensity of the peak may indicate the amount of the portion of the substance at the position of the peak. In some embodiments, the position of the peak is calculated against a neutral reference standard (e.g, DMSO) being characterized by a mobility peak at 0, and a charged reference standard (e.g., benzylamine) being characterized by a mobility peak at 1.0. In some embodiments, a population of lipid nanoparticles may exhibit more than one peaks as measured, by CZE, and unless indicated, otherwise, the mobility peak refers to the peak having the greatest peak area among the more than one peaks.
[0812] The term “free of,” as used herein, means not comprising the referenced component. For example, when a population, solution, or formulation is described as being “free of PEG lipid,” the population, solution, or formulation does not comprise PEG lipid (e.g, does not comprise a PEG lipid described herein (e.g, does not comprise PEG-DMG)).
[0813] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present, disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims. [0814] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a. group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process.
[0815] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used, herein, the terms “consisting essentially of” and “consisting of” are thus also encompassed and disclosed. Throughout the description, where compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
[0816] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary' skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0817] In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may' be excluded even if the exclusion is not set forth explicitly herein.
[0818] All cited sources, for example, references, publications, patent applications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.
[0819] The disclosure having been described, the following examples are offered by way of illustration and not limitation. EXAMPLES
[0820] It is understood that, unless specified otherwise, values presented in the examples are approximate, and are subject to experimental and instrumental variations..
Example 1: Synthesis of Exemplary Compounds [0821] General Procedure 1.
[0822] In a conical flask, modified Glc or GlcNAc substrate in DMSO and UDP-Glucose was dissolved in 0.2 M of Tris buffer at room temperature. 10 U of GalE was added into the reacti on mixture followed by 10 U of LgtB. Reaction mixture was incubated at room temperature for 48 h. Reaction was quenched by adding chilled ethanol and stirred for 30 min at room temperature. Later, reaction mixture was centrifuged at 1500xG for 10 min and the supernatant was decanted. The residue was diluted with water and centrifuged. The supernatant was collected and lyophilized with acetonitrile as a cosolvent. Resulting crude material was dissolved in water and purified by size exclusion chromatography (Sephadex G-15 resin) in water as an eluent. The desired fractions were collected and lyophilized with acetonitrile as a cosolvent. Resulting solid material was dissolved, in water and purified by reverse phase column chromatography in acetonitrile/water. The desired fractions were collected and lyophilized with acetonitrile as a cosolvent to afford lactose or LacNAc intermediate. [0823] In a conical flask NeuSAc and CTP were dissolved in 0.2 M of sodium cacodylate buffer at room temperature. 500 U of NmCSS were added into the reaction mixture at room temperature. Reaction mixture was incubated at 37 °C on an orbital shaker for 5 h. Then, lactose or LacNAc intermediate was added to the reaction followed by 5 U of a-2,6-sialyltransferase (Pd26ST). Reaction mixture was incubated at 37 °C on an orbital shaker for 48 h. Reaction was quenched by adding chilled ethanol and stirred for 30 min at room temperature. Later, reaction mixture was centrifuged at 1500xG for 10 min and the supernatant was decanted. The residue was diluted with water and centrifuged. The supernatant was collected and lyophilized with acetonitrile as a. cosolvent.. Resulting crude material was dissolved in water and purified by size exclusion chromatography (Sephadex G-15 resin) in water as an eluent. The desired fractions were collected and lyophilized with acetonitrile as a cosolvent. Resulting solid material was dissolved in water and purified by reverse phase column chromatography in acetonitrile/water. The desired fractions were collected and. lyophilized with acetonitrile as a cosolvent to afford, modified 2,6-sialyllactose product.
[0824] Buffer solution preparation:
[0825] Sodium cacodylate stock solution: Dissolve 53.50 g sodium cacodylate in 400 ml of water and adjust to pH 7.5 by adding 2 M HCL solution and dilute up to 500 ml with water.
[0826] Tris HCl Stock Solution; Dissolve 30.28 g Tris HCL in 400 ml of water and adjust to pH 7.5 by adding 2 M HCl solution and dilute up to 500 ml with water.
[0827] MgCb. stock solution; Dissolve 2.03 g of MnCl2 in 5 ml of water and dilute up to 10 ml. [0828] BSA stock solution; Dissolve 2 g of BSA in 10 ml of water and dilute up to 20 ml.
[0829] 0.2 M Tris buffer; Mix 80 ml of Tris HCl stock solution, 8 ml of MgCl2. stock solution and 2 ml of BSA stock solution and dilute up to 200 ml with water.
[0830] 0.2 M sodium cacodylate buffer; Mix 80 ml of sodium cacodylate stock solution, 8 ml of MgCI2 stock solution and 2 ml of BSA stock solution and diluted up to 200 ml with water.
[0831] General Procedure 2:
[0832] In a. conical flask, modified Glc or GlcNAc substrate in DMSO and UDP-Glucose was dissolved in 0.2 M of Tris buffer at room temperature. 10 U of GalE was added into the reaction mixture followed by 10 U of LgtB. Reaction mixture was incubated at room temperature for 48 h. Reaction was quenched by adding chilled ethanol and stirred for 30 min at room temperature. Later, reaction mixture was centrifuged at 1500xG for 10 min and the supernatant was decanted. The residue was diluted with water and centrifuged. The supernatant was collected and lyophilized with acetonitrile as a. cosolvent. Resulting crude material was dissolved in water and purified by size exclusion chromatography (Sephadex G-15 resin) in water as an eluent. The desired fractions were collected and lyophilized with acetonitrile as a cosolvent. Resulting solid material was dissolved in water and purified by reverse phase column chromatography in acetonitrile/water. The desired fractions were collected and. lyophilized with acetonitrile as a cosolvent to afford lactose or LacNAc intermediate.
[0833] In a conical flask Neu5Ac and CTP were dissolved in 0.2 M of sodium cacodylate buffer at room temperature. 500 U of NmCSS were added into the reaction mixture at room temperature. Reaction mixture was incubated at 37 °C on an orbital shaker for 5 h. Then, lactose or LacNAc intermediate was added to the reaction followed by 5 U of a-2,3-sialyltransferase (PmSTl). Reaction mixture was incubated, at 37 °C on an orbital shaker for 48 h. Reaction was quenched by adding chilled ethanol and stirred for 30 min at room temperature. Later, reaction mixture was centrifuged at 1500xG for 10 min and the supernatant was decanted. The residue was diluted with water and centrifuged. The supernatant was collected and lyophilized with acetonitrile as a. cosolvent. Resulting crude material was dissolved in water and purified by size exclusion chromatography (Sephadex G-15 resin) in water as an eluent. The desired fractions were collected and lyophilized with acetonitrile as a cosolvent. Resulting solid material was dissolved in water and purified by reverse phase column chromatography in acetonitrile/water. The desired fractions were collected and lyophilized with acetonitrile as a. cosolvent to afford modified 2,3-sialyllactose product.
[0834] General Procedure 3;
[0835] In a reaction flask, modified sialyllactose substrate and palladium on carbon were dissolved in methanol at room temperature. Reaction was sparged with hydrogen for 30 min and filtered through a pad of celite and washed with methanol. Solvent was removed under reduced pressure and resulting deprotected amine compound was carried, to the next step without purification. In a reaction flask, the crude amine compound and DSPE-PEG-NHS compound, was dissolved in DMF at room temperature. Then, diisopropylethylamine was added to the reaction and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was purified by directly injecting to preparative HPLC The desired fractions were collected and. lyophilized with acetonitrile to afford glycan modified PEG lipid, product.
Synthesis of Exemplary Intermediates
[0836] Acetic anhydride was added dropwise at 0 °C to a stirred solution of 1 in pyridine followed by DMAP. The reaction mixture was stirred at room temperature for 16 h. Upon completion, the reaction was cooled to 0 °C and quenched by adding water. The reaction was extracted with EtOAc and combined organic layers were washed with IM HC1 solution and brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. Resulting acylated product was carried to the next step without further purification. Crude reaction mixture was dissolved in DCM and was added HBr in AcOH dropwise at 0 °C. Reaction mixture was stirred at room temperature for 4 h. The reaction mixture was quenched, with saturated aqueous sodium bicarbonate and extracted with DCM. Combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to afford 2 which was used for next step without further purification.
[0837] To a solution of 2 and 3 in DCM was added NaH2PO4 at room temperature and stirred at for 10 min. Silver triflate was added at 0 °C. Reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filter through a pad of celite and washed with DCM. The filtrate was washed with 1M HC1 followed by saturated aqueous sodium bicarbonate, water, and brine. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. Resulting crude was purified by column chromatography to afford acetal intermediate. Then, to a solution of purified material in methanol was added sodium methoxide at 0 °C and reaction mixture was stirred at room temperature for 4 h. Amberlist- 15 (acid resin) was added portion wise up to pH 4-5. The acid resin was removed, by filtration and filtrate was evaporated under reduced pressure. Resulting crude material was purified by preparative HPLC to afford 4 as a white solid.
[0838] Compound 4: 1H NMR (400 MHz, DMSO): δ 7.38-7/23 (m, 5H), 5.10-5.04 (m, 2H),
[0839] TMSOTf was added dropwise to a stirred solution of 5 in DCE and the reaction mixture was stirred at 55 °C for 12 h. Reaction was cooled to 0 °C and added excess triethylamine. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with DCM, dried over sodium sulfate and concentrated under reduced pressure. The crude product was carried to the next, step without further purification.
[0840] Molecular sieves 4Å was added to a stirred, solution of the crude product and. 7 in DCE and stirred for 30 min at room temperature. Then, the reaction mixture was cooled to 0 °C and TMSOTf was added dropwise, then stirred at room temperature for 18 h. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with DCM, dried over sodium sulfate and concentrated under reduced pressure. The crude was purified by flash column chromatography to afford acetal intermediate as a colorless liquid.
[0841] To a stirred solution of the acetal intermediate in methanol at 0 °C was added sodium methoxide and reaction mixture was stirred at room temperature for 5 h. Them, Amberlist-15 (acid resin) was added portion wise up to pH 4-5. The acid resin was removed by filtration and solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC to afford 8 as a white solid.
[0842] Compound 8: 1H NMR (400 MHz, DMSO): δ 7.66 (d, J 8.4 Hz, 1H), 7.38-7.22 (m, 6H), 4.99 (s, 2H), 4.96 (d, J = 4 Hz, IH), 4.88 (d, J = 5.6 Hz, 1H), 4.51 (t, J = 5.8 Hz, IH), 4.24 (d, J = 8.4 Hz, 1H), 3.71-3.64 (m, 2H), 3.44-3.37 (m, 2H), 3.29-3.26 (m, 1H), 3.04 (s, 2H), 2.99-2.94 (m, 2H), 1.77 (s, 3H), 1.42-1.35 (m, 4H), 1.23 (s, 4H). MS: m/z = 455.52 [M+H] +
[0843] Molecular sieves 4A was added to a stirred solution of 6 and 9 in DCE and stirred at room temperature for 30 min. Then, the reaction was cooled to 0 °C and TMSOTf was added dropwise, then stirred for 18 h at room temperature. The reaction mixture was quenched with saturated aqueous NaHCOg and extracted, with DCM, dried over sodium sulfate and. concentrated under reduced pressure. The crude was purified by flash column chromatography to afford acetal intermediate as a colorless liquid.
[0844] To a stirred solution of the acetal intermediate in methanol at 0 °C was added sodium methoxide and reaction mixture was stirred at room temperature for 4 h. Then, Amberlist-15 (acid resin) was added portion wise up to the pH 4-5. The acid resin was removed by filtration and solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC to afford 10 as a white solid.
[0845] Compound 10: 1H NMR (400 MHz, DMSO): δ 7.67 (d, J - 8.8 Hz, 1H), 7.38-7.27 (m, 6H), 5.00 (s, 3H), 4.92 (d, J = 4.8 Hz, IH), 4.53 (t, J = 5.6 Hz, 1H), 4.31 (d, ,/ = 8.0 Hz, 1H), 3.78 (t, J =5.2 Hz, 1H), 3.67 (dd, J = 6.4, 5.6 Hz, 1 H), 3.52-3.36 (m, 7H), 3.30 (d, J 6.8 Hz, 1H), 3.13 (d, ./ 5.6 Hz, 2H), 3.06 (m, 211 ), 1.78 (s, 311). MS: m/z - 443.33 [M+H]+
[0846] Molecular sieves 4Å was added to a stirred solution of 6 and 11 in DCE and stirred at room temperature for 30 min. Then, the reaction was cooled to 0 °C and TMSOTf was added dropwise, then stirred for 18 h at room temperature. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted, with DCM, dried over sodium sulfate and. concentrated under reduced pressure. The crude was purified by flash column chromatography to afford acetal intermediate as a colorless liquid.
[0847] To a stirred solution of the acetal intermediate in methanol at 0 °C was added sodium methoxide and reaction mixture was stirred at. room temperature for 4 h. Then, Amberlist-15 (acid resin) was added portion wise up to the pH 4-5. The acid resin was removed by filtration and solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC to afford .12 as a white solid.
[0848] Compound 12: 1H NMR (400 MHz, DMSO): δ 7.68 (d, J - 8.8 Hz, 1H), 7.83-7.30 (m, 5H), 7.12 (t, J = 5.2 Hz, IH), 4.99 (s, 2H), 4.97 (cl, J = 4.0 Hz, 1H), 4.88 (d, ,/ = 5.2 Hz, 1H), 4.51 (t, J = 5.8 Hz, IH), 4,24 (d, J = 8.4 Hz, IH), 3.78 (t, J = 7.6 Hz, IH), 3.67 (dd, J = 6.0, 5.6 Hz, 1H), 3.46-3.37 (m, 3H), 3.26 (t, ./ 6.8 Hz, IH), 3.05 (s, 211), 2.97 (p, J 4.2 Hz, 2H), 1.77 (s, 3H), 1.42-1.30 (m, 4H), 0.84 (s, 6H). MS: m/z = 469.56 [M+H] +
[0849] To a stirred solution of 13 in water was added sodium hydroxide at 0 °C and stirred for 30 min. Then phthalic anhydride was added and stirred for 18 h at room temperature. Solvent was evaporated and. the crude mixture was dissolved in pyridine. Acetic anhydride was added at 0 °C and stirred for another 18 h at room temperature. Solvent was evaporated and the crude mixture was dissolved in DCM. Then the solution was washed with 1 N HC1 solution, saturated aqueous NaHCOg solution and brine, then dried on sodium sulfate and concentrated under reduced pressure. The crude mixture was purified by column chromatography to afford phthalate intermediate as a white solid.
[0850] To a stirred solution of the phthalate intermediate in DCM was cooled to 0 °C and HBr in acetic acid was added dropwise and stirred for 3h. Reaction was washed with saturated aqueous NaHCO3 solution and brine. The organic layer was dried over sodium sulfate and concentrated under reduced pressure to afford 14 as an off-white solid. The crude mixture was carried to the next step without further purification.
[0851] To a stirred solution of 14 and 15 in toluene was refluxed at 95 °C under nitrogen atmosphere. Two portions of AIBN was added over 2 h and the reaction mixture was stirred under reflux for 18 h. .After cooled to room temperature, the reaction mixture was diluted with diethyl ether and 10% aqueous KF solution. The biphasic mixture was vigorously stirred at room temperature for 24 h. The white solid was filter off and filtrate vras again diluted with diethyl ether, washed with water and brine. The organic layer was dried on sodium sulfate and. concentrate in vacuo. The crude was purified by column chromatography to afford allylated product as white solid.
[0852] To a stirred solution of the allylated product and 17 in DCM was added Grubbs’ 2nd gen. catalyst. The reaction mixture was stirred at 40 °C for 18 h. Then, solvent was evaporated under reduced pressure and the crude mixture was purified by column chromatography to afford 18 as an off-white solid.
[0853] Ta a stirred solution of 18 in IPA:H2O (6: 1) was added NaBH4 at 0 °C and stirred for 18 h at room temperature. Then, pH was adjusted to 4-5 by adding acetic acid and the reaction mixture was stirred under reflux for 8 h. Then, the reaction was cooled to room temperature and solvent was evaporated under reduced pressure. The crude reaction mixture was dissolved in pyridine and was added acetic anhydride at 0 °C. Catalytic amount of DM AP was added and the reaction mixture was stirred for 18 h at room temperature. Then, solvent was removed under reduced pressure. The crude material was dissolved in EtOAc, washed with 10% HC1 solution, saturated aqueous NaHCO3 solution and brine. The organic layer was dried over sodium sulfate and concentrate in vacuo. Resulting solid was triturated with pentane and dried in vacuum to obtain 19 as a white solid and carried to the next step without further purification.
[0854] To a stirred solution of compound 19 in MeOH was added Pd/C at room temperature. The reaction mixture was stirred for 18 h under H2 atmosphere. Then, Pd/C was filtered through a pad of celite and solvent was evaporated in vacuo. Benzyl chloroformate was added drop wise to the crude mixture at 0 °C and. the reaction mixture was stirred for 18 h at room temperature. Then, solvent was evaporated in vacuo. The crude mixture was purified by column chromatography to afford Cbz-protected intermediate as an off-white solid.
[0855] To a stirred solution of the Cbz-protected intermediate in methanol at 0 °C was added sodium methoxide and reaction mixture was stirred at room temperature for 4 h. Then, Amberlist- 15 (acid resin) was added portion wise up to the pH 4-5. The acid, resin was removed by filtration and solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC to afford 20 as a white solid.
[0856] Compound 20: 1H NMR (400 MHz, DMSO): δ 7.59 (d, J 9.2 Hz, 1H), 7.38-7.22 (m, 6H), 5.00 (s, 2H), 4.90 (d, J = 4.4 Hz, 1H), 4.76 (d, J = 5.2 Hz, IH), 4.38 (t, J= 5.8 Hz, 1H), 3.65 (dd, J--- 5.2, 5.6 Hz, 1H), 3.43-3.36 (m, 2H), 3.21-3.15 (m, 1H), 3.06-2.94 (m, 5H ), 1.81 (s, 3H), 1.45-1.38 (m, 4H), 1.21 (bs, 8H). MS: m/z = 453.48 [M+H]+
[0857] N-acetyl-D-glucosamine 21 in water was added NH4HCO3 until saturation and the solution was stirred at 50 °C. NH4HCO3 solid was continuously added to the reaction to maintain saturation. The reaction mixture was stirred at 50 °C for 48 h. Then, solvent was evaporated under reduced pressure. The crude mixture wwaass purified by column chromatography to afford amine intermediate as a white solid.
[0858] To a stirred solution of the amine intermediate in DMF at 0 °C was added HOBt, EDC- HC1 and DIPEA. The reaction mixture was stirred at 0 °C for 30 min and 22 was added. Then, the reaction mixture was stirred at room temperature for 18 h. Solvent was removed under reduced pressure and the crude mixture was purified by preparative HPLC to afford 23 as a white solid.
[0859] Compound 23: 1H NMR (400 MHz, DMSO): δ 7.98 (d, J = 9.2 Hz, 1 H), 7.81 (d, J = 8.4 Hz, 1H), 7.38-7.23 (m, 6H), 4.99 (s, 2H), 4.96 (d, J= 4.8 Hz, 2H), 4.79 (t, J= 9.4 Hz, 1H), 4.53 (t, J = 5.4 Hz, IH), 3.65 (dd, J = 5.2, 4.8 Hz, 1H), 3.51 (m, 1 H), 3.05 (m, 2H), 2.95(m, 2H), 2.07-1.99 (m, 2H), 1.77 (s, 3H), 1.42-1.35 (m, 4H), 1.81 (m, 2H). MS: m/z = 468.39 [M+H] +
[0860] N-acetyl-D-glucosamine 21 in water was added NH4HCO3 until saturation and the solution was stirred at 50 °C. NH4HCO3 solid was continuously added to the reaction to maintain saturation. The reaction mixture was stirred at 50 °C for 48 h. Then, solvent was evaporated under reduced pressure. The crude mixture was purified by column chromatography to afford amine intermediate as a white solid.
[0861] To a stirred solution of the amine intermediate in DMF at room temperature was added bis(2,5-dioxopyrrolidin-1-yl) carbonate and the reaction mixture was stirred for 4 h. Then, 24 in DMF was added dropwise at 0 °C. The reaction mixture was stirred for another 18 h at room temperature. Solvent was removed under reduced pressure and the crude mixture was purified, by preparative HPLC to afford 25 as a white solid.
[0862] Compound 25: 1H NMR (400 MHz, DMSO): δ 7.84 (d, J = 8.8 Hz, 1H), 7.38-7.26 (m, 6H), 6.32 (s, 1H), 6.05 (d, J = 9.2 Hz, H i) 4.99 (s, 2H), 4.94 (d, J = 4.8 Hz, 1H), 4.90 (d, J = 5.6 Hz, 1H), 4.63 ft, ,/ = 9.6 Hz, 1H), 4.51 (t, J = 5.8 Hz, 1H), 3.61 (dd, J = 5.2, 4.8 Hz, H I). 3.51-3.40 (m, 2H), 3.25 (dd, J = 9.6, 7.2 Hz, 1H), 3.11-2.94 (m, 6H), 1.80 (s, 31 1). 1.34 (t, ,7= 3.6 Hz, 4H). MS: m/z = 469.35 [M+H]+
[0863] To a stirred solution of 5 was added acetyl chloride at room temperature. The reaction mixture was stirred at room temperature for 24 h. Then, reaction mixture was concentrated under reduced pressure to afford chloride intermediate as a colorless oil. The crude mixture was carried to the next step without further purification.
[0864] To a stirred solution of the chloride intermediate in acetone was added thiourea at room temperature. The reaction mixture was stirred at 60 °C for 2 h. Then, precipitated solid was filter out and wash with acetone and vacuum dried to obtain crude thiourea intermediate as an off-white solid. The crude mixture was carried to the next step without further purification.
[0865] To a. stirred solution of the thiourea, intermediate in DCM and waster was added sodium metabisulfide at room temperature. The reaction mixture was refluxed for 3 h. Then, reaction mixture was cooled to room temperature and the mixture was extracted with DCM, dried on sodium sulfate and concentrated to obtain crude 26 as an off-white solid. The erode mixture was carried to the next step without further purification.
[0866] DBU was added in a solution of 26 and 27 in MeCN and stirred for 4 h at room temperature. Then, solvent was removed under reduced pressure. Resulting mixture was dissolved in water and extracted with DCM, dried on sodium sulfate and vacuum dried. The crude mixture was purified by column chromatography to afford alkylated intermediate as a white solid. The crude mixture was carried to the next step without further purification.
[0867] At 0 °C, TFA was added in a solution of the alkylated intermediate in DCM and stirred for 2 h at room temperature. Then, solvent was evaporated and crude mixture was washed, with diethyl ether to obtain 28 as a white solid. The crude mixture was carried to the next step without further purification.
[0868] To the solution for 28 in DCM was added triethylamine followed by benzyl chlorofomiate at room temperature. The reaction mixture was stirred at room temperature for 5 h. Then, the reaction was quenched with water, extracted with DCM, dried over sodium sulfate and concentrated under reduced pressure. The erode mixture was purified by column chromatography to afford sulfide intermediate as a white solid.
[0869] To a stirred solution of the sulfide intermediate in methanol at 0 °C was added sodium methoxide and reaction mixture was stirred at room temperature for 4 h. Then, Amberlist-15 (acid, resin) was added portion wise up to the pH 4-5. The acid, resin was removed by filtration and solvent was evaporated under reduced pressure. The crude mixture was washed with 1 : 1 diethyl ether/ pentane, dissolved in water and acetonitrile and lyophilize to afford 29 as a white solid.
[0870] Compound 29: 1H NMR (400 MHz, DMSO): δ 7.69 (d, J = 9.2 Hz, 1H), 7.38-7.28 (m, 51 1). 7.23 (t, J = 5.6 Hz, 1H), 4.99 (s, 3H), 4.96 (d, J = 5.2 Hz, 1 H), 4.51 (t, J = 5.8 Hz, 1H), 4.30 (d, J = 10.8 Hz, 1H), 3.66 (dd, J= 5.6 Hz, 1H), 3.51-3.40 (m, 2H), 3.24 (d, J = 5.6 Hz, 1H), 3.07 (s, 2H), 2.96 (q, J = 6.26 Hz, 2H), 2.96 (q, J = 6.1.3 Hz, 2H), 178 (s, 31 1) 1.48 (t, J = 6.4 Hz, 2H), 1.37 (t, J = 6.8 Hz, 2H), 1.29-1.23 (m, 4H). MS: m/z = 470.76 [M+H]+
Synthesis of Examplary Sialic Acid Lipids:
[0871] 30 was synthesized, from 4 fallowing General Procedure 1.
[0872] Compound 30: 1H NMR [400 MHz, D2O]: δ == 7 22(b, 5H), 4.90(s, 2H), 4.26 (d, .1 8 Hz, IH), 4,21 (d, 1=8 Hz, 1H), 3.77-3.30(m, 20 H), 3.14-3.09(m, 1 H), 2.91 (bs, 2H), 2.52(dd, J =4 Hz, 12 Hz, 1H), 1.82(s, 3H), 1.54 (t, J 12 Hz. 1 H) 1.40-1.13(m, 8H). MS: m/z 865.56[M-H]‘
[0873] 31 was synthesized from 30 following General Procedure 3.
[0874] Compound 31 (Compound No. 1) : 1H NMR [400 MHz, MeOD]: δ = 5.22 (s, IH),
4,44 (dd, J - 12.0, 3.2 Hz, IH), 4.35 - 4.27 (m, IH), 4.26 - 4. 15 (m, 3H), 4.08 3.96 (m , 5H), 3.96 -• 3.78 (m, 8H), 3.63 (d, J = 3.6 Hz, 167H), 3.59 - 3.38 (m, 10H), 3.19 - 3.13 (m, 2H),
2.73 (d, J = 12.4 Hz, IH), 2.66 - 2.58 (m, 2H), 2.48 (q, J = 8.8 Hz, 2H), 2.33 (dt, J 11.2, 7/4
Hz, 4H), 1.99 (d, J =3.5 Hz, 2H), 1.76 (d, J = 6.9 Hz, IH), 1.61 (dt, J = 14.3, 6.9 Hz, 6H), 1.51 (d, J = 8.3 Hz, 2H), 1.30 (d, J = 10.6 Hz, 56H), 0.90 (t, J = 6.9 Hz, 6H). PDI (Mw/Mn, GPC)= 1.01
[0875] 34 was synthesized from 8 following General Procedure 1.
[0876] Compound 34: NMR [400 MHz, D2O]: 5 - 7.38-7,37 (m, 5H), 5.06 (s, 2H), 4,49(d, J =7:2 Hz, 1H), 4.39(d, >1:2 Hz, 1H), 4.20-3 ,49(m, 20 H), 3.070 (t, 3H), 2.62 (dd, >4.4, 12.2 Hz 1H), 1.99(s, 6H), 1.67(1; J = 2.4 Hz, 1H) 1.55-1.43 (m ; 4H), 1 .25 (m, 4H) MS: m/z = 907.42 [M+H] +
[0877] 35 was synthesized from 34 following General Procedure 3.
[0878] Compound 35 (Compound No. 2): 1H NMR [400 MHz, MeOD]: δ = 5.22 (s, 1H), 4.57 (d, J 8.4 Hz, 1H), 4.45 (dd, ./ 12.1, 3.2 Hz, H I). 4.33 (d, J 7.5 Hz, 1 H), 4.20 (dq, J = 18.4, 5.7 Hz, 3H), 4.09 (t, J= 9.3 Hz, 1H), 4.03 - 3.95 (m, 4H), 3.95 - 3.79 (m, 7H), 3.64 (d, J = 1.6 Hz, 177H), 3.58 - 3.39 (m , 11H), 3.27 - 3.11 On. 4H), 2.63 (t, J -- 7.1 Hz, 2H), 2.48 (t., J= 7.0 Hz, 2H), 2.33 (dt, J= 10.8, 7.3 Hz, 4H), 1.99 (d, J= 5.7 Hz, 5H), 1.77 (t, J= 12.2 Hz,
1H1 1.66 1.45 On.. 8H), 1.30 (d. ./ 10.5 Hz, 69H), 0.90 (t, J = 7.0 Hz, 6H). PDI (Mw/Mn,
GPC) 1.01
[0879] 36 was synthesized from 10 following General Procedure 1.
[0880] Compound 36: 1H NMR [400 MHz, D2O]: δ - 7.39 (m, 5H), 5.06 (s, 2H), 4.54(d, J-7.4 Hz, 1H), 4.39(d, .N7.2 Hz, 1H), 3.95-3.49(m,27 H), 3.27 (s, 2H), 2.62 (dd, J = 4.5, 12.2 Hz 1H), 1.99-1.98(s, 6H), 1.67(1, ,7=12.4 Hz,1 H). MS: m/z = 896.53 [M+H]
[0881] 37 was synthesized from 36 following General Procedure 3.
[0882] Compound 37 (Compound No. 3): 1H NMR [400 MHz, MeOD]: δ = 5.22 (dd, J = 6.7, 3.5 Hz, IH), 4.64 (d, J = 8.3 Hz, IH), 4.45 (dd, J = 12.1, 3.2 Hz, 1H), 4.33 (d, J = 7.6 Hz, 1H), 4.27 -- 4.14 (m, 3H), 4.08 (t, J = 9.3 Hz, IH), 4.04 - 3.88 (m, 8H), 3.88 -- 3.75 (m, 5H), 3.64 (d, J =- 2.5 Hz, 182H), 3.56 - 3.41 (m, 10H), 2,72 (dd, J = 11.7, 3.9 Hz, 1H), 2.67 - 2.58 (m, 2H), 2.57 -- 2.47 (m, 2H), 2.33 (dt, J= 11.3, 7.5 Hz, 4H), 2.00 (d, J= 8.2 Hz, 4H), 1 .61 (d, J = 7.1 Hz, 4H), 1.29 (s, 67H), 0.90 (1, -J 6.9 Hz, 6H). PDI (Mw/Mn, GPC) - 1.01
[0883] 38 was synthesized from 12 following General Procedure 1.
[0884] Compound 38: 1H NMR [400 MHz, D2O]: δ = 7.40-7.39 (m, 5H), 5.08 (s, 2H), 4.52(bs, 1H), 4.41(d, J = 8 Hz, 1H), 3.99-3.77(m, 10 H), 3.68-3.51 (m, 13H), 3.10 (t, J = 8.0 Hz, 2H), 2.64 (dd, .7=4,3, 12.1 Hz IH), 2.00(s, 6H), 1.69(t, J =12.0 Hz, IH), 1.58-1.37 (m, 4H), 0.87 (s, 6H). MS: m/z - 922.70 [M+H]+
[0885] 39 was synthesized from 38 following General Procedure 3.
[0886] Compound 39 (Compound No. 4): 1H NMR [400 MHz, MeOD]: δ = 5.22 (d, J= 6.7 Hz, 1H), 4.55 (d, J = 8.3 Hz, IH), 4.45 (dd, J = 12.0, 3.2 Hz, 1H), 4.33 (d, J = 7.6 Hz, 1H), 4.26 - 4. 15 (m, 3H), 4.10 (t, J= 9.4 Hz, IH), 4.06 - 3.78 (m, 12H), 3.64 (s, 172H), 3.56 - 3.38 (m, 9H), 3.25 - 3.15 (m, 4H), 2.66 (dq, J 38.2, 6.4 Hz, 3H), 2.48 (q, J 8.7 Hz, 2H), 2.33 (dt, J= 11.9, 7.4 Hz, 4H), 1.99 (d, J= 8. 1 Hz, 5H), 1.80 (t, J= 12.2 Hz, 1H), 1.58 (dq, J= 27.0, 7.1 Hz, 5H), 1.30 (s, 551 1 ). 0.94 (s, 5H), 0.91 (t, ./ 6.9 Hz, 6H). PD1 (MwZMn, GPC) - 1 .01
[0887] 40 was synthesized from 20 following General Procedure 1.
[0888] Compound 40: 1H NMR [400 MHz, D2O]: δ - 7.39(m, 5H), 5.08 (s, 2H), 4.41(d, ,7=7.6 Hz, 1H), 3.99-3.48 (m, 19H), 3.36 (bs, 1H), 3.08 (bs, 2H), 2.64 (dd, ,7=4.5, 12.3 Hz 1H), 2.02-2.00 (s, 6H), 1.70 (t, ,7=12.3 Hz, 1H) 1.69-1.54 (m, 5H), 1.53-1.21 (m, 7H). MS: m/z = 903.53 [M-1] +
[0889] 41 was synthesized from 40 following General Procedure 3.
[0890] Compound 41 (Compound No. 5): 1H NMR [400 MHz, MeOD]: δ = 44..9933 - 4.87 (m, 1H), 4.12 (dd, ,7 = 12.0, 3.2 Hz, 1H), 3.98 (dd, ,7= 22.7, 6.1 Hz, 1H), 3.95 -- 3.82 (m, 3H), 3.77 - 3.46 (m, 11H), 3.31 (s, 182H), 3.23 - 3.07 (m, 8H), 2.92 - 2.80 (m, 8H), 2.43 - 2.36 (m, 1H), 2.30 (t, ,/ = 7.0 Hz, 2H), 2. 15 (t, J = 7.0 Hz, 2H), 2.00 (dt, J = 11.0, 7.4 Hz, 4H), 1 .67 (d, ,/ = 13.9 Hz, 5H), 1.45 - 1.36 (m, 1H), 1.31 - 1.20 (m, 5H), 1.19 - 1.12 (m, 2H), 1.10 - 0.91 (m, 70H), 0.58 (t, J= 7.0 Hz, 6H). PDl (Mw/Mu, GPC) = 1 .01 [0891] 42 was synthesized from 23 following General Procedure 1.
[0892] Compound 42: 1H NMR [400 MHz, D2O]: δ = 7.39(m, 5H), 5.07-5.05 (bs, 3H), 4.42(4. ,7=8.0 Hz, H I), 3.96-3.74 (m, I OH), 3.68-3.48 (m, 10H), 3.07 (bs, 2H), 2.65 (dd, .7=4.6, 12.4 Hz 1H), 2.23 (bs, 2H), 1.98 (s, 6H), 1.71 (t, 7=12.4 Hz, 1H) 1.56-1.43 (m, 4H), 1.25-1.23 (m, 2H). MS: m/z = 919.58 [M+H] +
[0893] 43 was synthesized from 42 following General Procedure 3.
[0894] Compound 43 (Compound No. 6): 1H NMR [400 MHz, MeOD]: δ 4.89 (dd, J ----- 6.9, 3.6 Hz, 1H), 4.71 (d, .7 = 8.8 Hz, 1H), 4.12 (dd, J= 12.0, 3.2 Hz, 1H), 3.99 (dd, J = 30.2, 6.1 Hz, 1H), 3.94 - 3.82 (m, 3H), 3.76 - 3.46 (m, 10H), 3.31 (s, 178H), 3.25 - 3.04 (m, 8H), 2.92 -- 2.77 (m, 4H), 2.45 - 2.38 (m, H I). 2.35 - 2.25 (m, 211). 2.15 (t, J 7.0 Hz, 211). 2.00 (dt, J = 11.1, 7.3 Hz, 4H), 1.87 (td, J= 7.4, 3.1 Hz, 1H), 1.70 - 1.63 (m, 4H), 1.38 (d, J= 7.6 Hz, 1H), 1 .28 (q, J 7.4 Hz, 5H), 1.18 (t, J 7.6 Hz, 2H), 0.97 (q, J 6.7 Hz, 58H), 0.58 (t, J = 6.9 Hz, 6H). PDI (Mw./Mn, GPC) = 1.01
[0895] 44 was synthesized from 25 following General Procedure 1.
[0896] Compound 44: *H NMR [400 MHz, D2O]: δ = 7.40 (m, 5H), 5.07 (s, 2H), 4.90(d, 7=8.4 Hz, H I ), 4.41 (d, ,7=8.0 Hz, H I). 3.99-3.75 (m, 1 H i). 3.68-3.51 (m, 10H), 3.07 (bs, 4H), 2.64 (dd, ,7=4.4, 12.4 Hz 1H), 1.99 (s, 6H), 1.69 (t, ,7=12.4 Hz, 1H) 1.43 (m, 4H). MS: m/z = 919.54 [M+H] +
[0897] 45 was synthesized from 44 following General Procedure 3.
[0898] Compound 45 (Compound No. 7): 1H NMR [400 MHz, MeOD]: δ = 5.22 (s, IH), 4.44 (dd, J 11.8, 3.2 Hz, IH), 4.31 (dd, J 21.1, 6.1 Hz, IH), 4.20 (kid, J --- 16.2, 9.8, 6.3 Hz, 3H), 4.09 -- 3.90 (m, 6H), 3.90 - 3.79 (m, 4H), 3.64 (d, J 2.2 Hz, 189H), 3.56 -- 3.40 (m, 7H), 3/23 - 3.06 (m, 5H), 2.77 - 2,54 (m, 4H), 2.49 (d, ,/ = 7.0 Hz, IH), 2.39 - 2.28 (m, 4H), 2.00 (d, J --- 11.3 Hz, 4H), 1.71 (t, J --- 12.0 Hz, I H), 1.61 (q, J 7.3 Hz, 4H), 1.55 - 1.45 (m, 3H), 1.31 (dd, J = 14.5, 5.2 Hz, 59H), 0.90 (td, J= 7.0, 2.2 Hz, 6H). PDI (Mw/Mn, GPC) = 1.01
[0899] 46 was synthesized, from 29 following General Procedure 1.
[0900] Compound 46: 1H NMR [400 MHz, D2O]: δ == 7.38 (m, 5H), 5.07 (s, 2H), 4.59(d, ,7=10 Hz, IH), 4.40(d, ,7=8.0 Hz, IH), 3.98-3.50 (m, 21H), 3.07 (bs, 2H), 2.70-2.61 (m, 3H), 2.01 ( s, 3H), 1.98 (s, 3H), 1.67 (t, 7=12 Hz, IH) 1.61-1.27 (m, 8H). MS: m/z ------ 924.43 [M/-H]+
[0901] 47 was synthesized from 46 following General Procedure 3. [0902] Compound 47 (Compound No. 8): 1H NMR [400 MHz, MeOD]: δ - 5.22 (dd, .J 6.8, 3.7 Hz, 1H), 4.52 (d, J = 10.4 Hz, 1H), 4.44 (dt, J= 12.1, 3.3 Hz, 1H), 4.39 - 4.25 (m, 1H), 4.25 - 4.11 (m, 3H), 4.1 1 - 3.80 (m, 10H), 3.80 - 3.57 (m, 180H), 3.57 - 3.38 (m, 7H), 3.26 - 3.13 (m, 7H), 2.67 - 2.54 (m, 3H), 2.49 (dd, J = 7.1, 3.3 Hz, 2H), 2.33 (dt, J= 11.2, 7.4 Hz, 5H), 2.06 - 1 .91 (m, 3H), 1 .86 - 1 .66 (m, 2H), 1 .65 - 1 .46 (m, 7H), 1 .36 - 1.22 (m, 63H), 0.94
- 0.85 (m, 6H). PDI (Mw/Mn, GPC) = 1.01
[0903] 48 was synthesized from 4 following General Procedure 2.
[0904] Compound 48: 1H NMR [400 MHz, D2O]: δ = 7.41-7.39 (m, 5H), 5.07 (s, 2H), 4.49 (d, J 8 Hz, 1H) 4.42 (d, J 8 Hz,lH), 4.07 (dd J 8.4 Hz, 2.4 Hz, 1H), 3.95-3.49 (m, 16H), 3.31-3.22 (m, 1H), 3.08 (bs, 2H), 1.82(s, 3H), 2.74-2.70 (dd, J=4 Hz, 12.4 Hz, 1H) 1.99 (s, 3 H), 1.79-1.73(1, >12.4 Hz, lH), 1.57-1.30(m, 8H). MS: m/z - 865.59 [M-H]'
[0905] 49 was synthesized, from 48 following General Procedure 3.
[0906] Compound 49 (Compound No. 9): 1H NMR [400 MHz, MeOD]: δ 5.22 (s, 1H),
4.48 4.40 (m, 2H), 4.26 - 4.16 (m, 3H), 4.08 - 3.96 (m, 5H), 3.96 - 3.80 (m, 7H), 3.63 (d, J - 6.1 Hz, 175H), 3.59 - 3.38 (m, 9H), 3.27 - 3.13 (rn, 3H), 2.83 (d. .7 .12.7 Hz, 1H), 2.62 (td, J= 7.7, 3.7 Hz, 2H), 2.49 (td, J= 6.9, 3.0 Hz, 2H), 2.33 (dt, .7= 1.1.3, 7.1 Hz, 4H), 2.00 (d, J = 6.6 Hz, 2H), 1.79 (d, J = 13.8 Hz, 1H), 1.61 (qd, J = 13.1, 6.7 Hz, 6H), .1.55 - 1.47 (m, 2H), 1.29 (d, J= 6.1 Hz, 57H), 0.91 (t, J= 6.7 Hz, 6H). PDI (Mw/Mn, GPC) = 1.01
[0907] 52 was synthesized, from 8 fallowing General Procedure 2.
[0908] Compound 52: 1H NMR [400 MHz, D2O]: δ = 7.38-7.37 (m, 5H), 5.06 (s, 2H), 4.54(d, ,7=8.2 Hz, IH), 4.48(d, J=7.9 Hz, 1H), 4.12-3.50(m,20 H), 3.10 (t, 3H), 2.74 (dd, 7=4.4, 12.2 Hz, Hz 1H), 2.02-1.99(s, 6H), 1.79(t, 7=12,4 Hz, 1H) 1.51-1.46 (rn, 4H), 1.28 (m, 4H), MS: m/z = 907.45 [M+H] +
[0909] 53 was synthesized from 52 following General Procedure 3.
[0910] Compound 53 (Compound No. 10): 1H NMR [400 MHz, MeOD]: δ 5.26 5.19
(m, 1H), 4.50 -- 4.42 (m, 2H), 4.38 (d, J = 8.3 Hz, IH), 4.27 - 4.14 (m, 3H), 4.09 - 3.80 (m, 1 IH), 3.64 (d, J = 3.9 Hz, 177H), 3.54 - 3.37 (rn, 7H), 3.27 - 3.10 (m, 3H), 2.87 - 2.79 (m, 1H), 2.66 - 2.58 (m, 2H), 2.48 (dd, J = 9.2, 4.8 Hz, 2H), 2.33 (dt, J = 11.1, 7.4 Hz, 4H), 1.98 (d, J = 27.0 Hz, 4H), 1.82 - 1.73 (rn, IH), 1.67 - 1.45 (m, 7H), 1.30 (s, 57H), 0.90 (t, J 6.8 Hz, 6H). PDI (Mw/Mn, GPC) = 1.01
[0911] 54 was synthesized from 10 following General Procedure 2. [0912] Compound 54: 1H NMR [400 MHz, D2O]: δ = 7.41 (m, 5H), 5.10 (s, 2H), 4.52-4.47 (m, 3H), 4.08 (d, ,7=9.6 Hz, 1H), 3.95-3.77(m, 9H), 3.70-3.51 (m, 13H), 3.29 (s, 3H), 2.73 (dd, .7=4,2, 12.3 Hz H I), 2.00-1.97(s, 6H), 1 .77(1.. 7 12.4 Hz, H I) MS: m/z = 893.69 [M-H]"
[0913] 55 was synthesized from 54 following General Procedure 3.
[0914] Compound 55 (Compound No. 11): 1H NMR [400 MHz, MeOD]: δ = 5.22 (s, 1H), 4.45 (q, 7 7.4 Hz, 2H), 4.26 - 4.15 (m, 3H), 4.07 - 3.82 (m, 121 h. 3.78 -- .3.55 (m, 181H), 3.51 (dt, 7 = 20.2, 6.7 Hz, 5H), 2.82 (d, 7 = 12.6 Hz, 1H), 2.64 (t, 7 = 7.1 Hz, 2H), 2.53 (t, 7 = 7.1 Hz, 2H), 1.99 (dt, 7 = 20.4, 1.9 Hz, 51 1). 1.61 (q, ./ 7.2 Hz, 4H), 1.30 (d, 7 10.8 Hz, 59H), 0.94 -- 0.86 (m, 6H). PDI (Mw/Mn, GPC) = 1.01
[0915] 56 was synthesized from 12 following general procedure 2.
[0916] Compound 56: 1H NMR [400 MHz, D2O]: δ = 7.39-7.38 (m, 5H), 5.07 (s, 2H), 4.52- 4.47 (m, 2H), 4.07(m, 1H), 3.96-3.74(m, 8H), 3.68-3.51 (m, 13H), 3.09 (t, J= 8.0 Hz, 2H), 2.72 (dd, 7=4.4, 12.2 Hz 1H), 1.99(s, 3H), 1.96(s, 3H), 1 .76(1 , 7=12.2 Hz, 1H), 1.47-1.35 (m, 411), 0.85 (s, 6H). MS: m/z = 922.57 [M+H]: [0917] 57 was synthesized from 56 following General Procedure 3.
[0918] Compound 57 (Compound No. 12): 1H NMR [400 MHz, MeOD]: δ 5.25 5.19
(m, IH), 4.48 - 4.41 (m, I H), 4.38 (d, .7 = 8.6 Hz, IH), 4.27 - 4.15 (m, 3H), 4.07 - 3.80 (m, 10H), 3.64 (s, 167H), 3.55 - 3.38 (m, 7H), 3.25 - 3.14 (m, 3H), 2.85 (dd, J= 12.3, 3.7 Hz, IH), 2.67 - 2.58 (m, 2H), 2.48 (t, J = 7.0 Hz, 21 1), 2.33 (dt, J = 10.8, 7.4 Hz, 4H), 1 .99 (dd, ./ 28.9, 2.3 Hz, 4H), 1.74 (t, ,7 = 11.5 Hz, IH), 1.58 (dq, ,7 = 30.5, 8.2 Hz, 5H), 1.30 (s, 62H), 0.96 - 0.85 (m, 10H). PDI (Mw/Mn, GPC) = 1.01
[0919] 58 was synthesized from 20 following General Procedure 2.
[0920] Compound 58: 1H NMR [400 MHz, D2O]: δ = 7.40(m, 5H), 5.08 (s, 2H), 4.52(d, ,7=8.0 Hz, IH), 4.09 (d, ,7=8.8 Hz, IH), 3.95-3.52 (m, 16H), 3.46-334 (m, 3H), 3.08 (bs, 211), 2.73(dd, ,7=4.4, 12.1 Hz IH), 2.00-1.99 (s, 6H), 1.78 (t, ,7=12.1 Hz, IH) 1.69-1.54 (m, 5H), 1.53-1.21 (m, 7H). MS: m/z = 903.53 [M-H]"
[0921] 59 was synthesized from 58 following General Procedure 3.
[0922] Compound 59 (Compound No. 13): 1H NMR [400 MHz, MeOD]: δ = 4.89 (dt, .7 = 5.8, 3.0 Hz, IH), 4.16 - 4.09 (m, 2H), 3.93 - 3.81 (m, 3H), 3.74 - 3.63 (m, 5H), 3.63 - 3.47 (m, 7H), 3.31 (d, ,7= 3.4 Hz, 18711), 3.27 - 3.12 (m, 8H), 2.92 - 2.74 (m, 10H). 2.51 (dd, J
12.5, 3.6 Hz, IH), 2.30 (t, J = 7.0 Hz, 2H), 2.15 (t, J = 7.0 Hz, 2H), 2.00 (dt., J = 11.3, 7.4 Hz, 41 1), 1.66 (dd, .7= 23.1, 3.4 Hz, 51 1), 1.33 - 1.11 (m, 81 1), 1.11 - 0.90 (m, 741 1), 0.58 (t, .7= 6.9 Hz, 6H). PDi (Mw/Mn, GPC) = 1.01
[0923] 60 was synthesized from 23 following General Procedure 2.
[0924] Compound 60: 1H NMR [400 MHz, D2O]: δ - 7.42(rn, 5H), 5.09 (bs, 3H), 4.55 (bs, IH), 4, I0(d, J 9.2 Hz, 1H), 3.94-3.56 (m, 18H), 3.10 (bs, 2H), 2.75 (dd, ./ 4.4, 12.4 Hz, I H), 2.23 (bs, 2H), 2.02-1.96 (s, 6H), 1.79 (t, ,7=12.4 Hz, IH) 1.55-1.47 (m, 4H), 1.26 (m, 2H). MS: m/z == 919.60 [M-H]-
[0925] 61 was synthesized from 60 following General Procedure 3.
[0926] Compound 61 (Compound No. 14): 1H NMR [400 MHz, MeOD]: δ = 5.22 (s, IH), 4.97 (d, J= 9.8 Hz, IH), 4.46 (ddd, J= 15.9, 8.0, 3.4 Hz, 2H), 4.20 (dq, J= 16.3, 5.8 Hz, 3H), 4.08 - 3.78 (m, 12H), 3.78 - 3.54 (m, 184H), 3.54 - 3.38 (m, 6H), 3.25 - 3.10 (m, 9H), 2.89 - 2.81 (m, 1 H), 2.62 (q, J --- 5.5 Hz, 2H), 2.51 -• 2.42 (m, 2H), 2.33 (di, J 11.3, 7.4 Hz, 4H), 2.19 (t, J= 7.5 Hz, 2H), 1.98 (dd, J= 32.3, 2.5 Hz, 5H), 1.75 (s, 1H), 1.66 - 1.53 (m, 6H), 1.50 (q, ./ 7.5 Hz, 2H), 1.41 - 1 .20 (m, 6611), 0.95 - 0.86 (m. 6H). PDI (Mw/Mn, GPC) == 1 .01
[0927] 62 was synthesized from 25 following General Procedure 2. [0928] Compound 62: 1H NMR [400 MHz, D2O]: δ = 7.39 (m, 5H), 5.07 (s, 2H), 4.53(bs, IH), 4.08(m, IH), 3.93-3.51 (m, 21H), 3.08 (bs, 4H), 2.73 (dd, 7=4.4, 12.4 Hz IH), 1.98 (s, 6H), 1.77 (t, 7 12.4 Hz, IH) 1.43 (m, 4H). MS: m/z - 919.57 [M-H]'
[0929] 63 was synthesized from 62 following General Procedure 3.
[0930] Compound 63 (Compound No. 15): 1H NMR [400 MHz, MeOD]: 5 = 4.89 (s, 1H), 4.25 (s, IH), 4. 16 - 4.07 (m, 2H), 3.93 - 3.80 (m, 3H), 3.75 - 3.45 (m, I IH), 3.30 (d, J = 8.2 Hz, 178H), 3.21 - 3.06 (m, 7H), 2.92 - 2.72 (m, 9H), 2.58 -- 2.48 (m, IH), 2.33 -- 2.25 (m, 2H), 2.14 (dt., J ------ 10.9, 4.3 Hz, 2H), 2.00 (de ./ 11.1, 7.4 Hz, 4H), 1.65 (dd, ./ 24 8, 8.5 Hz, 4H), 1.40 (d, .7 = 11.5 Hz, IH), 1.27 (t, J= 7.4 Hz, 4H), 1.20 - 1.11 (m, 3H), 0.97 (q, J = 8.2 Hz,
61H), 0.58 (t, J= 7.3 Hz, 6H). PDI (Mw/Mn, GPC) 1.01
[0931] 64 was synthesized from 29 following General Procedure 2.
[0932] Compound 64: *H NMR [400 MHz, D2O]: δ = 7.38 (bs, 5H), 5.07 (s, 2H), 4.57- 4.57(m, 2H), 4.40(d, 7 8 0 Hz, IH), 4.08(d, ./ 10 Hz, I H), 3.95-3.51 (m, 19H), 3.08 (bs, 211), 2.73-2.63 (m, 3H), 1.99( s, 3H), 1.98 (s, 3H), 1.76 (t, 7=12 Hz, IH) 1.54-1.25 (m, 8H). MS: m/z - 924.43 [M+H] +
[0933] 65 was synthesized from 64 folkwing General Procedure 3.
[0934] Compound 65 (Compound No. 16): 1H NMR [400 MHz, MeOD]: δ 5.25 5.19
(m, 1H), 4.49 - 4.40 (m, 2H), 4.20 (ddd, ./ 15.8, 8.7, 5.3 Hz, 3H), 4.08 - 3.80 (m, 10H), 3.64
(d, ./ = 1.1 Hz, 170H), 3.54 - 3.37 (m, 6H), 3.25 - 3.11 (m, 8H), 2.84 (dd, J = 12.6, 3.5 Hz, H E), 2.77 - 2.57 (m, 31 1) 2.48 (t, J ------ 7.0 Hz, 1H), 2.33 (dt, J ------ 1 .1.0, 7.4 Hz, 4H), 2.03 1.94
(m, 4H), 1.76 (t, J = 11.6 Hz, 1H), 1.61 (q, ,/= 7.2 Hz, 5H), 1.44 - 1.18 (m, 62H), 0.90 (t, J
6.9 Hz, 5H). PDI (Mw/Mn, GPC) - 1.01
Example 2. Description of PIPA Process to Manufacture LNPs
[0935] Sialic acid (Compound 1 and Compound 9) LNPs were manufactured with two variations of Post insertion, post addition (PIPA) process. In one process, sialic acid lipid was incorporated in the nanoprecipitation stage (FIG. 1). The lipid mixture, consisting of the ionizable lipid, DSPC, cholesterol, and 0.25-1% of sialic acid lipid, were dissolved in ethanol at 12.5mM ( 4.25 mL/min) and brought into contact with an aqueous stream (pH 5, 25mM acetate, : 12.75 mL/min) containing the mRNA. The two streams were mixed at a volumetric ratio of 1 :3 (lipids:RNA) and the RNA concentration was adjusted for an N/P ratio of 4.9. The two streams were mixed using a 0.3mm Poseidon mixer with a small-scale setup to mimic the process used at scale. The mixed product (25% ethanol) was diluted 3x by volume, using pH 6, lx Citrate Buffered Saline (CBS, 51 mL/min) to lower the ethanol content to 6.25%. The LNPs were allowed, to mature at this stage for 30 mins. After this hold, the LNPs were brought into contact with either a Phosphate, Tris or HEPES buffer to raise the pH above 7 (see Table 1 for pH-adjust buffer composition). The volume of the pH adjust buffer was set at 5 w/v% of the mixed product. In order to stabilize the LNPs over tangential flow filtration (TFT), a post-insertion (PI) step was performed to raise the PEG content on the LNPs (see Table 2 for PI buffer composition). After PI, the LNPs were concentrated (>0.5 mg/mL) and exchanged into the final storage buffer (see Table 3 for final buffer composition). For a small-scale batch (- 1 - 3 mg), this buffer exchange is performed using Amicon filters and desalting columns, while at scale (-20 mg) TFF is typically used. After concentration and buffer exchange, a final post-addition (PA) of PEG to the LNPs was performed to raise the PEG content to the target molar composition.
[0936] In the second variation of the process, the introduction of sialic acid lipids occurs at a different stage (FIG. 2). Here, the lipid components, which include the regular four components, are dissolved in ethanol and then mixed with the aqueous stream that carries the mRNA. Instead of incorporating the sialic acid lipids in the lipid stock solution, in this process, they are dissolved in the post-insertion (PI) buffer along with additional PEG and added after the neutralization step. The remainder of the procedure aligns with that, of the first version.
[0937] HPLC analysis indicated that sialic acids were incorporated as part of the LNP (see FIG. 3). to o o
+
Example 3: Delivery to Hematopoietic Stem and Progenitor Cells and Erythroid Progenitor Cells
[0938] The interaction of lipid nanoparticles of the present disclosure with hematopoietic stem and progenitor cell subpopulations within the bone marrow was evaluated. Table 6 provides the percent of cells targeted in bone marrow. Table 7 provides expression of green lantern protein in cells. Table 8 provides the shift in stem cell antigen- 1 (Sea-1) expression, which is a surrogate marker of inflammatory reaction.
BM flow cytometry
[0939] Mice were sacrificed through CO2 asphyxiation followed by centical dislocation. Humeri, tibias, femurs and hips were collected and crashed in a 100 mm dish using a 50 ml. conical in 10 mLs phosphate Buffered Saline (PBS) without calcium or magnesium supplemented with 2% Fetal Bovine serum (PBS + ) For the hematopoietic stem and progenitor cell (HSPC) panel (see Table 9), cells were strained using a 70 μm strainer and subjected to red blood cell (RBC) lysis using the Easy Sep™ Red Blood Cell Lysis Buffer (StemCell Technologies™) for 5 minutes, after whi ch 5 mLs of PBS+ were added and cells were spun at 500g and supernatant discarded. Cells were re-suspended in PBS+, and nucleated cells were counted using Acridine Orange. Twenty million cells were blocked in fluorescence active cell sorting (FACS) buffer (PBS+ with 0.5 mM EDTA [Ethylenediaminetetraacetic acid]) containing 1 :50 Normal rat serum for 10 minutes at room temperature. An equal volume of 2X staining mix was added (staining at 40 Million cells/mL), cells were mixed and incubated at 4
°C for 30 minutes and then washed using FACS buffer and resuspended in FACS buffer containing the viability dye 7- Aminoactinomycin D (7AAD) and analyzed using the BD
FAC SymphonyTM S6. For the erythroid progenitor cell (EPC) panel (see Table 10), cells were not subjected to RBC lysis and 10 Million cells were stained and analyzed similar to the HSPC panel. Data was analyzed using FlowJo. Data, are presented in Tables 6-8. Taken together, the data in this Example demonstrate that LNPs comprising Compound 1 are particularly effective at delivering mRNA to EPCs and HSPCs. Example 4: Delivery to myeloid and lymphoid ceils
Spleen flow cytometry
[0940] Eighteen hours after intravenous mRNA LNP delivery , mice were sacrificed through CO2 asphyxiation followed by cervical dislocation. Spleens were collected and crushed in a 6- well plate in RPMI medium with 10% Fetal Bovine Serum (FBS). Cells were strained using a 70 μm strainer and subjected to 1 mL RBC lysis using ACK lysis buffer (Thermo Fisher Scientific®) for 2 minutes, after which 1 ml, of PBS with 2% FBS and 1 mM EDTA (PBSFE) was added and cells were spun at 500g and supernatant discarded. Cells were re-suspended in PBSFE and nucleated cells counted. Two million cells were stained with Viability Live Dead BLUE (Thermo Fisher Scientific) and Fc-blocked for 30 minutes on ice. Cells were then washed with PBSFE and resuspended in FACS buffer containing an antibody panel (see Table 12). Samples were fixed and data collected on the SONY ID7000 flow cytometer and analyzed with OMIQ. Data are presented in Table 11. Taken together, the data in this Example demonstrate that LNPs comprising Compound 1 are effective at delivering mRNA to myeloid and lymphoid cells in the spleen.
ls>
CD
Example 5: Delivery of gene editing systems to hematopoietic stem cells in vivo
[0941] Lipid nanoparticles of the disclosure were used for in vivo delivery spCas9 mRNA and a sgRNA that, included a targeting sequence directed to mouse CD33 molecule (CD33), and the % of insertions/deletions (indels) in CD33 were measured.
[0942] Wild type C57ZB16 mice were dosed intravenously with co-form dated spCas9 mRNA and an sgRNA with a targeting sequence against mouse CD33 (at a 1 ; 1 mass ratio), as indicated in FIG. 5, with or without hematopoietic stem cell mobilization (M). Mobilization was done using Neupogen (125 μg/Kg/Day) for four days in 2 split doses/day followed by a 5 mg/Kg
Plerixafor dose. LNPs were dosed 60 minutes after Plerixafor. Mice were euthanized 72 hours post LNP dosing and bone marrow hematopoietic stem and progenitor cells were enriched using the EasySep™ Mouse Hematopoietic Progenitor Cell Isolation kit (Ref#19856) and. Easy Sep TM Mouse CD117 Positive Selection kit (Cat#18 /57) from StemCell Technologies according to manufacturer’s instructions. DNA was extracted using the Maxwell RSC Tissue DNA Kit from Promega (Ref#AS1610) and indels were determined using Amplicon Sequencing. Data in FIG. 5 is presented as mean +/- standard deviation. Statistical significance was calculated using OneWay ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. The sequences of the spCas9 protein and sgRNA are shown in Table 13 below. Taken together, the data in this example demonstrate that LNPs comprising Compound I are effective at delivering mRNA. encoding gene editing systems to hematopoietic stem cells in vivo.
Table 13. Sequences of gene editing constructs
Example 6: Delivery of sialic add containing LNP to human cells as demonstrated nsing humanized mice
[0943] Humanized female mice (Hu-CD34+NSG-SGM3) were purchased from the Jackson Laboratory. These are stock# 013062, which are engrafted with human cord blood CD34+ cells. Engrafted human CD34+ cells in these mice also express CD33, and CD34 and not CD33 was used for FACS analysis, as described below. Mice were dosed intravenously at 0.5 mg/Kg with the indicated green lantern mRNA LNP formulations and euthanized 24 hours post dosing. Bone marrow (BM) was collected from one hindlimb by crushing the bones and then subjecting the BM to red blood cell lysis using the EasySep Red Blood Cell lysis buffer (StemCell TechnologiesTM) according to manufacturer’s instructions. Cells were then stained for flow cytometry at 40 million cells/mL using the panel in Table 14 for 30 minutes at 4 °C and then were washed and analyzed using the BD FACSymphony S6. My eloid progenitors are hCD45+, Lineage negative, CD34+ and. CD38+ (FIG. 6A). Hematopoietic stem and progenitor cells (HSPCs) are hCD45+, Lineage negative, CD34+ and CD38- (FIG. 6B). Long-term repopulating hematopoietic stem cells (LT-HSCs) are 11CD45+, Lineage negative, CD34+, CD38-, and CD45RA- (FIG. 6C).
[0944] The percentage of cells expressing green lantern for each cell type, and with each of the indicated green lantern LNP formulations, are shown in FIGS. 6A-6C. Data is presented as mean +/-standard deviation and statistical significance is calculated using one-way ANOVA, * p<0.05, ** p<0,01, *** p<0.001, **** p< 0001. Taken together, the data in this example demonstrate that LNPs comprising Compound 1 are effective at delivering mRNA to human LT-HSCs, HSPCs and myeloid progenitors.
Example 7: Immunogenicity of sialic acid lipid nanopartities
[0945] In orderto determine if sialic acid lipid nanoparticles can reduce inflammatory' cytokine responses while maintaining immunogenicity, the cytokine profiles were measured upon administration of sialic acid lipid mtnoparticles indicated in Table 15 was assayed.
[0946] BALB/c mice (55 total) were intramuscularly administered mRNA encoding influenza hemagglutinin (HA) protein using sialic acid LNP of the indicated, formulation and dose in Table 15, at the times indicated in FIG. 7. Serum was collected on the first day (6 hours after dosing), on day 21, on day 22 (6 hours after administering a booster dose), and on day 36. On day' 36, mice were euthanized, and the spleens were also collected for analysis.
209 [0947] Following euthanization, mouse spleens were collected and stored in RPMI10 media (Roswell Park Memorial Institute 1640 medium with ATCC modification, supplemented with L-Glutamine, Penicillin/Streptomycin, and 10% heat-inactivated fetal bovine serum [FBS]). Single-cell suspensions were prepared from BALB/c mouse spleens using a gentleMACS tissue dissociator. After tissue dissociation, red blood cell lysis was performed for 5 minutes, followed by quenching with RPMI10 media, and filtration through a 70 μm filter. Cells from each mouse were counted using a Cellaca MX system (FIG. 9), resuspended in RPMI10 media, and incubated at 37°C with 5% CO2 for 6 hours in the presence of Protein Transport Inhibitor, 2 μg/mL of 15-amino-acid peptides overlapping by 11 amino acids encoding Flu A/Wisconsin HA1 and. CD107a PE. Control conditions included DMSO (0.5% final, negative control) and. PMA/Ionomycin (positive control).
[0948] After incubation, cells were washed with PBS and stained with LIVE/DEAD Fixable LTV Blue Dead Cell Stain for 15 minutes at room temperature. Cells were then washed with FC stain buffer (PBS supplemented with 2% heat-inactivated FBS and 0.05% sodium azide). Surface staining was performed with a cocktail of antibodies prepared in Brilliant Stain Buffer, including FC Block, CD4 BV480, CD44 BUV395, I-A/I-E AF488, and CD8 BUV805. The staining cocktail was added to the cells and incubated for 15 minutes at room temperature. Cells were then washed with FC stain buffer, fixed, and permeabilized using the BD Cytofix/Cy toperm kit according to the manufacturer’s instructions. After permeabilization, cells were washed with IX Perm/Wash Buffer. Average cell viability is shown in FIG 8.
[0949] Intracellular staining was performed at 4 °C for 20 minutes using a cocktail of antibodies in Brilliant Stain Buffer, including CD3 BV605, IFN-γ BV421, TNF-α BV711, IL- 5 APC, IL-4 PE-CF594, IL-13 PE-Cy7, and IL-2 BV785. Finally, cells were washed, with 1X Perm/Wash Buffer and FC stain buffer, filtered through a 30 μm filter in a 96-well plate, and resuspended in 0.5% PFA-FC stain buffer. Samples were acquired on an Aurora Spectral Flow Cytometer (Cytek. Biosciences). T cell intracellular cytokine staining (ICS) analysis was subsequently performed using OMIQ. Lymphocytes were gated from the total population on an FSC-A vs. SSC-A plot, followed by doublet exclusion using a singlet gate on an FSC-A vs. FSC-H plot. Dead cells were excluded using a Live/Dead Blue vs. SSC-A plot. CD34MHCII cells were gated from the live, single-cell population on an MHCII (IA-IE) vs. CD3 bivariate plot to exclude antigen-presenting cells (APCs). CD44 and CDS4 T cell populations were separated on a CDS vs. CD4 bivariate plot. Cytokine expression (Th1: IFNγ, TNFα, IL-2, or Th2: IL-4, IL-5, and/or IL-13) and the degranulation marker CD107a (CDS* T cells only) were assessed within the CD4+ and CD8+ populations via co-expression with the T cell activation marker CD44 on a CD44 vs. Cytokine bivariate plot. Background cytokine expression from the no-peptide control condition (DMSO) was subtracted from that measured in peptide- stimulated samples for each individual mouse. Values < 0.005 were thresholded to 0.0025, which represents a lower limit of detection (LLOD) that is 2-fold lower than the lowest observed positive signal.
[0950] The results are shown in FIGS, 10-11 . FIG. 10 show the CD4 T cell response in groups 1 , 3, 5, 7 and 9 from Table 15. All groups had lowered Th2 cytokine responses relative to group 3. Group 5 showed lower overall T-cell responses compared to Group 3. Groups 7 and 9 and showed similar T-cell responses to control Group 3. FIG. 11 shows the CD8 T cells response in groups 1, 3, 5, 7 and 9 from Table 15. Group 5 showed lower overall CD8 T-cell responses compared to Group 3. Groups 7 and 9 showed more robust CDS T-cell responses relative to control Group 3.
[0951] The hemagglutination inhibition (HAI) assay was performed to measure receptor- blocking antibody titers against influenza virus A/Sydney/5/2021. Serum samples were pre- treated with receptor-destroying enzyme (RDE) (Denka Seiken, cat # 370013) to remove nonspecific inhibitors by mixing one part serum with three parts RDE, incubating at 37°C for 18 hours, and inactivating the enzyme at 56°C for 30 minutes. Treated sera were diluted 1: 10 with phosphate-buffered saline (PBS) before use. Fresh red blood cells (RBCs) from guinea pig (Lampire, cat # 7243108) were washed three times with lx PBS and resuspended to a final concentration of 0.75%. A/Sydney/5/2021 was diluted, to 8 hemagglutination units (HAU) per 50 μL in lx PBS. Serial two-told dilutions of RDE-treated serum were prepared in 96-well U- bottorn microtiter plates with lx PBS as the diluent. Each well received 25 μL of diluted serum and 25 μL of virus, followed by incubation at room temperature for 30 minutes with shaking. Subsequently, 50 μL , of 0.75% guinea pig RBC suspension was added, and. plates were incubated at room temperature for 60 minutes to observe hemagglutination patterns. The endpoint titer was defined as the highest serum dilution that completely inhibited hemagglutination, recorded, as the reciprocal of this dilution. Data were analyzed using GraphPad Prism.
[0952] Total IgG titers to recombinant H1 A/Sydney/5/2021 protein, as measured ELISA, are shown in FIG. 13. Groups 6-7 showed improved titers relative to Groups 2-3 in both the low and high dose groups. All animals in the high dose Group 7 seroconvert by day 21. Groups 4- 5 and Groups 8-9 showed reduced antibody responses relative to Groups 2-3 at day 36 and both dose groups. [0953] Cytokine responses 6 hours post boost were assayed using a Luminex immunoassay. The results for all groups are shown in FIGS. 14-15. Overall, cytokine responses were low as expected due to the low dose used to discriminate immunogenicity effects. IFNg showed a marked reduction from high-dose Group 3 with all other LNPs, though two individuals were driving the overall response. CXCL1 was highest with Groups 2-3, and showed reduction for all other groups. TNFa showed a reduction from high dose Group 3, with all other LNPs. CCL2 showed substantial reduction from Groups 2-3 with all other LNPs. IFNa. and IL-12 were near or below LOD for all groups. CXCL10 showed a marked reduction from high-dose Group 3 with all other LNPs. IL-6 is highest with Groups 2-3 and showed a reduction for all other groups. CCL5, IL- lb, and IL-10 were trending with Groups 2-3 as highest over other groups. [0954] GM-CSF was near or below LOD for all groups.
[0955] The hemagglutination inhibition antibody responses of sera, from mice from the indicated treatment group are shown in FIG. 12. Groups 6-7 improved HAI-active antibody titers at both dosages tested relative to Groups 2-3. Groups 4-5 and 8-9 caused a reduction in HAI titers relative to Groups 2-3 at both dosages tested. All constructs elicited responses over background (PBS, Group 1).
[0956] Taken together, the data in this Example demonstrate that LNPs comprising Compound 9 maintain levels of Thl cytokines and T cells, which are known to be important in promoting neutralizing antibody responses, while reducing Th2 cytokine responses, which have been shown to be detrimental to the development of productive antiviral immune responses. This has been shown for both CD4+ T cells and CD8+ T cells. LNPs comprising Compound 9 increase both binding and functional antibody titers, while reducing the overall inflammatory cytokine profile after intramuscular administration. In summary, these LNPs improve the quality of immune responses while reducing inflammatory responses.
EQUIVALENTS
[0957] The details of one or more embodiments of the invention are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.
[0958] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the invention to the precise form disclosed, but by the claims appended hereto.

Claims

WHAT IS CLAIMED IS:
1. A sialic acid lipid of Formula (SA-I): or a salt or ionized form thereof, wherein:
M is ; wherein * indicates attachment to R;
R is C13-20 alkyl or C13-20 alkenyl;
M’ is wherein * indicates attachment to R’;
R’ is C 13-20 alkyl or C13-20 alkenyl;
X+ is a. pharmaceutically acceptable cation, n is 40-50;
L is -(C3-8 alkylene)-!-* or -(C3-8 heteroalkylene)-!-*, wherein the C3-8 alkylene or C3- 8 heteroalkylene is optionally substituted with one or more oxo;
T is
-La is , wherein indicates attachment to L and indicates attachment to Sa;
R1 is -OR1" or -NRr"-C(=0)-Rr’";
R1 is H or C1-6 alkyl;
Rr is H or C1-6 alkyl;
R3 ’ is C1-6 alkyl; and
-Sa. is
2. The sialic acid lipid of claim 1, wherein the lipid is of Formula (SA-II): or a salt or ionized form thereof.
3. The sialic acid lipid of claim 1 or 2, wherein the lipid is of Formula (SA-II”): or a salt or ionized form thereof.
4. The sialic acid lipid of claim 1 or 2, wherein the lipid is of Formula (SA-II”): or a salt or ionized form thereof.
5. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-III):
or a salt or ionized form thereof.
6. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-IV): or a salt or ionized form thereof.
7. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-V): (SA-V) or a salt or ionized form thereof.
8. The sialic acid lipid, of any one of the previous claims, wherein the lipid is of Formula (SA-V-i): or a salt or ionized form thereof.
9. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-V-ii): or a salt or ionized form thereof.
10. The sialic acid lipid of any one of the previous claims, wherein the lipid Is of Formula (SA- VI): or a salt or ionized form thereof.
11. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-VI-i): or a salt or ionized form thereof.
12. The sialic acid lipid of any one of the previous claims, wherein the lipid is of Formula (SA-VI-ii): or a salt or ionized form thereof.
13. The compound of any one of the previous claims, wherein R is a C13, C15, or C17 alkyl.
14. The compound of any one of the previous claims, wherein R’ is a C13, C15, or C17 alkyl.
15. The compound of any one of the previous claims, wherein n is 42 to 44.
16. The compound of any one of the previous claims, wherein M is *-C(=O)-O-, wherein
* indicates attachment to R
17. The compound of any one of the previous claims, wherein M’ is *-C(=O)-O-, wherein
* indicates attachment to R.
18. The compound of any one of the previous claims, wherein X+ is a sodium cation.
19. The compound of any one of the previous claims, wherein L is , , wherein indicates attachment to -La-Sa.
20. The compound of any one of the previous claims, wherein -La is , wherein indicates attachment to L and indicates attachment to -Sa.
21. The compound of any one of the previous claims, wherein -La is wherein indicates attachment to L and indicates attachment to -Sa.
22. The compound one of the previous claims, wherein -La is wherein indicates attachment to L and indicates attachment to -Sa.
23. The compound of any one of the previous claims, wherein -La is wherein indicates attachment to L and indicates attachment to -Sa.
24. The compound of any one of the previous claims, wherein -Sa is
25. A lipid nanoparticle comprising a sialic acid lipid of any one of the previous claims.
26. The lipid nanoparticle of any one of the previous claims, further comprising an ionizable lipid, and a structural lipid.
27. The lipid nanoparticle of any one of the previous claims, further comprising a phospholipid.
28. A population of lipid nanoparticles comprising a sialic acid lipid of any one of the previous claims.
29. The population of lipid nanoparticles of any one of the previous claims, further comprising an ionizable lipid and a structural lipid.
30. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, comprising about 30 mol % to about 50 mol %, about 30 mol % to about 45 mol %, about 30 mol % to about 40 mol %, about 35 mol % to about 45 mol %, or about 35 mol % to about 40 mol % of the ionizable lipid.
31. The lipid nanoparticle or population of lipid nanoparticles of any one of the preceding claims, comprising about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, or about 50 mol % of the ionizable lipid.
32. The lipid nanoparticle or population of lipid, nanoparticles of any one of the preceding claims, wherein the ionizable lipid is compound 1-18 or Compound II-6.
33. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, comprising about 15 mol % to about 45 mol %, about 20 mol % to about 45 mol %, about 25 mol % to about 45 mol %, about 30 mol % to about 45 mol %, about 35 mol % to about 45 mol %, or about 40 mol % to about 45 mol % of the structural lipid.
34. The lipid nanoparticle or population of lipid nanoparticles of any one of the preceding claims, comprising about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, or about 45 mol % of the structural lipid.
35. The lipid nanoparticle or population of lipid nanoparticles of any one of the preceding claims, wherein the structural lipid is cholesterol.
36. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, comprising about. 10 mol % to about 30 mol %, 10 mol % to about 25 mol %, 10 mol % to about 20 mol %, about 15 mol % to about 25 mol %, or about 15 mol % to about 20 mol % of the phospholipid.
37. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims comprising about 10 mol %, about 15 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 25 mol %, or about 30 mol % of the phospholipid.
38. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, wherein the phospholipid is DMPS, DSPC, DOPE, DOPC, POPE, or POPC.
39. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, being free of PEG lipid.
40. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, further comprising a. PEG lipid.
41. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, comprising about 0.5 mol % to about 10 mol %, about 0.5 mol % to about 5 mol %, about 1 mol % to about 5 mol %, or about 1 mol % to about 3 mol % of the PEG lipid.
42. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, comprising about 0.5 mol %, about 1 mol %, about 2 mol %, about 2.5%, about. 3 mol %, about 4 mol %, or about 5 mol % of the PEG lipid.
43. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, wherein the PEG lipid is PL-02.
44. The lipid nanoparticle or population of lipid nanoparticles of any one of the preceding claims, comprising about 0.1 mol % to about 5 mol %, about 0.1 mol % to about 4 mol %, about 0.1 mol % to about 3 mol %, about 0.1 mol % to about 2 mol %, about 0.2 mol % to about 2 mol %, about 0.4 mol % to about 1.5 mol % of the sialic acid lipid, or about 0.4 mol% to about 1 mol% of the sialic acid lipid.
45. The lipid nanoparticle or population of lipid, nanoparticles of any one of the preceding claims, comprising about. 0.1 mol %, about 0.2 mol %, about 0.3 mol %, about 0.4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about. 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1 .6 mol %, about 1.7 mol %, about 1.8 mol %, about 1 .9 mol %, or about 2.0 mol % of the sialic acid lipid.
46. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, being free of therapeutic agent.
47. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, further comprising a therapeutic agent.
48. The lipid nanoparticle or the population of lipid nanoparticles of claim 47, wherein the therapeutic agent comprises a nucleic acid, optionally a RNA or a DNA, a protein, or a combination thereof.
49. The lipid nanoparticle or the population of lipid nanoparticles of claim XI , wherein the nucleic acid comprises a microRNA, shRNA, siRNA, or guide RNA for a CRISPR/Cas system.
50. The lipid nanoparticle or the population of lipid nanoparticles of any one of the preceding claims, further comprising a therapeutic agent wherein the therapeutic agent comprises a mRNA.
51. The lipid nanoparticle of claim 48, wherein the mRNA comprises an open reading frame encoding an antigen or a protein, optionally wherein the protein comprises a CRISPR protein.
52. The lipid nanoparticle of any one of the preceding claims, comprising:
(i) a sialic acid lipid of Formula (SA-1);
(ii) an ionizable lipid;
(iii) a structural lipid;
(iv) a phospholipid;
(v) a PEG lipid; and
(iv) a nucleic acid.
53. The lipid nanoparticle of any one of the preceding claims, comprising:
(i) a sialic acid lipid of Formula (SA-1); (ii) an ionizable lipid;
(iii) a structural lipid;
(iv) a phospholipid; and
(v) a PEG lipid; wherein the lipid nanoparticle is free of nucleic acid.
54. The lipid nanoparticle of claim 49 or 50, comprising:
(i) about 0.1 mol % to about 5 mol % of the sialic acid lipid;
(ii) about 30 mol % to about 50 mol % of the ionizable lipid;
(iii) about 5 mol % to about 45 mol % of the structural lipid;
(iv) about 10 mol % to about 30 mol % of the phospholipid; and
(v) about 0.5 mol % to about 10 mol % of the PEG lipid.
55. The lipid nanoparticle of any one of the preceding claims, comprising:
(i) a sialic acid lipid of Formula (SA-1);
(ii) an ionizable lipid;
(iii) a structural lipid;
(iv) a phospholipid;
(v) a PEG lipid; and
(iv) a nucleic acid; wherein the lipid nanoparticle comprises:
(i-a) about 0.1 mol % to about 5 mol % of the sialic acid, lipid;
(ii-a) about 30 mol % to about 50 mol % of ionizable lipid;
(iii-a) about 5 mol % to about 45 mol % of the structural lipid;
(iv-a) about 10 mol % to about 30 mol % of the phospholipid; and (v-a) about 0.5 mol % to about 10 mol % of the PEG lipid.
56. The lipid nanoparticle of any one of the preceding claims, comprising:
(i) a sialic acid lipid of Formula (SA-1),
(ii) an ionizable lipid;
(iii) a structural lipid;
(iv) a phospholipid; and
(v) a PEG lipid; wherein : the lipid nanoparticle is free of nucleic acid; and the lipid nanoparticle comprises:
(i-a) about 0.1 mol % to about 5 mol % of the sialic acid lipid, (ii-a) about 30 mol % to about 50 mol % of ionizable lipid;
(iii-a) about 5 mol % to about 45 mol % of the structural lipid, (iv-a) about 10 mol % to about 30 mol % of the phospholipid; and (v-a) about. 0.5 mol % to about 10 mol % of the PEG lipid.
57. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 0.2 mol% to about 2 mol% of sialic acid lipid.
58. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 0.5 mol% to about 1.5 mol% of sialic acid lipid and about 1.5 mol% to about 2.5 mol% of PEG lipid.
59. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 1 mol % sialic acid lipid and about 2 mol% PEG lipid.
60. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 45 mol % to about 55 mol % ionizable lipid.
61. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 35 mol % to about 45 mol % structural lipid.
62. The lipid nanoparticle of any one of the previous claims, wherein the lipid nanoparticle comprises about 8 mol % to about 12 mol % phospholipid.
63. The lipid nanoparticle of any one of the previous claims, wherein the sialic acid lipid is Compound 1 or Compound 9.
64. The lipid nanoparticle of any one of the previous claims, wherein the ionizable lipid is compound 1-18, 1-25, 1-301, II-6, or Vi-4.
65. The lipid nanoparticle of any one of the previous claims, wherein the phospholipid is DMPS, DSPC, DOPE, DOPC, POPE, or POPC.
66. The lipid, nanoparticle of any one of the previous claims, wherein the structural lipid is cholesterol .
67. The lipid nanoparticle of any one of the previous claims, wherein the PEG lipid is PEG- DMG or PL-02.
68. The lipid nanoparticle of any one of the previous claims, wherein the LNP comprises the combination of ionizable lipid, PEG-lipid, sialic acid lipid, and phospholipid as specified in Table LA”.
69. The lipid nanoparticle of any one of the previous claims, wherein: the ionizable lipid is 1-18; the PEG- lipid is PL-02; the structural lipid is cholesterol; the phospholipid is DSPC, and the sialic acid lipid is compound 1.
70. The lipid nanoparticle of any one of the previous claims, wherein: the sialic acid lipid is Compound 9 the ionizable lipid is 1-25; the structural lipid is cholesterol; the phospholipid is DSPC; and the PEG lipid is PL-02.
71. The lipid nanoparticle of any one of the previous claims, wherein the nucleic acid is mRNA.
72. A pharmaceutical composition comprising the lipid, nanoparticle or the population of lipid nanoparticles of any one of the preceding claims and one or more pharmaceutically acceptable carriers or excipients.
73. A method of delivering a. therapeutic agent to a. hematopoietic stem and progenitor cell (HSPC), an erythroid progenitor cell (EPC), a myeloid cell, or a lymphoid cell in a subject, comprising administering to the subject the lipid nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of any one of the preceding claims.
74. The lipid nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of any one of the previous claims for use in delivering a therapeutic agent to a hematopoietic stem and progenitor cell (HSPC), an erythroid progenitor cell (EPC), a myeloid cell, or a lymphoid cell in a subject.
75. Use of lipid, nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of any one of the previous claims in the manufacture of a medicament for delivering a therapeutic agent to a hematopoietic stem and. progenitor cell (HSPC), an erythroid progenitor cell (EPC), a myeloid cell, or a lymphoid cell in a subject.
76. The method, lipid nanoparticle, population, pharmaceutical composition, or use of any one of the preceding claims, wherein: the HSPC is in the spleen of the subject, or the HSPC is in the bone marrow of the subject.
77. The method, lipid nanoparticle, population, pharmaceutical composition, or use of any one of the preceding claims, wherein the lipid nanoparticle comprises Compound 1.
78. A method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the lipid nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of any one of the previous claims, wherein the lipid nanoparticle or population of lipid nanoparticles comprise a therapeutic agent.
79. The lipid nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of any one of the previous claims for use in treating or preventing a disease or disorder in a subject, wherein the lipid, nanoparticle or population of lipid nanoparticles comprise a. therapeutic agent.
80. Use of the lipid nanoparticle, the population of lipid nanoparticles, or the pharmaceutical composition of the previous claims in the manufacture of a medicament for treating or preventing a disease or disorder in a subject, wherein the lipid nanoparticle or population of lipid nanoparticles comprise a therapeutic agent.
81. The method, lipid nanoparticle, population, pharmaceutical composition, or use of any one of the previous claims, wherein: the subject is human; and/or the disease or disorder is a spleen disease or a spleen disorder, or the disease or disorder is a bone marrow disease or a bone marrow disorder.
A vaccine comprising a messenger ribonucleic (mRNA) acid formulated in the lipid nanoparticle of any one of any one of the previous claims, wherein the mRNA comprises an open reading frame encoding a. cancer antigen or an infectious disease antigen.
83. A vaccine comprising a messenger ribonucleic (mRNA) acid formulated in a lipid nanoparticle, wherein the mRNA comprises an open reading frame encoding a cancer antigen or an infectious disease antigen, wherein the lipid nanoparticle comprises:
(i) a sialic acid lipid of any one of claims 1-24;
(ii) an ionizable lipid;
(iii) a structural lipid;
(iv) a phospholipid; and
(v) a. PEG lipid.
84. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises:
(i) about 0.1 mol % to about 5 mol % of the sialic acid lipid;
(ii) about 30 mol % to about 50 mol % of the ionizable lipid;
(iii) about 5 mol % to about 45 mol % of the structural lipid,
(iv) about 10 mol % to about 30 mol % of the phospholipid; and
(v) about 0.5 mol % to about 10 mol % of the PEG lipid.
85. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises about 0.5 mol% to about 1.5 mol% of sialic acid lipid and about 1.5 mol% to about 2.5 mol% of PEG lipid.
86. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises about 1 mol% sialic acid lipid and about 2 mol% PEG lipid.
87. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises about 45 mol % to about 55 mol % ionizable lipid.
88. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises about 35 mol % to about 45 mol % structural lipid.
89. The vaccine of any one of the previous claims, wherein the lipid nanoparticle comprises about 8 mol % to about 12 mol % phospholipid.
90. The vaccine of any one of the previous claims, wherein the ionizable lipid is compound 1-18, 1-25, 1-301, II-6, or VI-4.
91. The vaccine of any one of the previous claims, wherein the phospholipid is DMPS, DSPC, DOPE, DOPC, POPE, or POPC.
92. The vaccine of any one of the previous claims, wherein the structural lipid is cholesterol.
93. The vaccine of any one of the previous claims, wherein the PEG lipid is PEG-DMG or PL-02.
94. The vaccine of any one of the previous claims, wherein the sialic acid lipid is compound 1 or compound 9.
95. The vaccine of any one of the previous claims, wherein the sialic acid lipid is compound
9.
96. The vaccine of any one of the previous claims, wherein: the sialic acid lipid is Compound 9 the ionizable lipid is 1-25; the structural lipid is cholesterol; the phospholipid is DSPC, and the PEG lipid is PL-02.
97. The vaccine of any one of the previous claims, wherein the mRMA comprises one or more modified nucleosides.
98. The vaccine of claim 82, wherein the one or more modified nucleosides comprise M l m ethyl -p seudouri di ne .
99. The vaccine of any one of any one of the previous claims, wherein the open reading frame comprises nucleosides consisting of N1 -methyl -pseudouridine, adenosine, guanosine, and cytidine.
100. The vaccine of any one of any one of the previous claims, wherein the infectiou s disease antigen is selected from the group consisting of a coronavirus antigen, an influenza vims antigen, a respiratory syncytial virus (RSV) antigen, a human metapneumovirus (hMPV) antigen, a cytomegalovirus (CMV) antigen, an Epstein-Barr virus (EBV) antigen, a herpes simplex vims (HSV) antigen, a varicella zoster virus (VZV) antigen, and a human immunodeficiency virus (HIV) antigen.
101. The vaccine of any one of the previous claims, wherein the infectious disease antigen comprises an influenza vims antigen, optionally wherein the influenza vims antigen comprises influenza, hemagglutinin 1 (Hl) or a portion thereof.
102. A method of reducing susceptibility to or eliminating symptoms of an infectious disease in a subject, comprising administering to the subject the vaccine of any one of the previous claims, wherein the mRNA comprises an open reading frame encoding an infectious disease antigen.
103. A method of treating cancer in a subject, comprising administering to the subject the vaccine of any one of the previous claims, wherein the mRNA comprises an open reading frame encoding a cancer antigen.
104. A composition comprising:
(a) nucleic acid comprising a sequence encoding a CRISPR nuclease; and/or
(b) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and
(c) the lipid nanoparticle of any one of the previous claims.
105. A composition comprising:
(a) nucleic acid comprising a sequence encoding a CRISPR nuclease;
(b) a guide RNA (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence; and
(c) a lipid nanoparticle comprising:
(c-i) an ionizable lipid,
(c-ii) a PEG lipid;
(c-iii) a structural lipid;
(c-iv) a phospholipid; and
(c-v) a sialic acid lipid of any one of claims 1-24.
106. The composition of claim 89 or 90, wherein the lipid nanoparticle comprises:
(i) about 0.1 mol % to about 5 mol % of the sialic acid lipid;
(ii) about 30 mol % to about 50 mol % of the ionizable lipid;
(iii) about 5 mol % to about 45 mol % of the structural lipid,
(iv) about 10 mol % to about 30 mol % of the phospholipid; and
(v) about 0.5 mol % to about 10 mol % of the PEG lipid.
107. The composition of any one of the previous claims, wherein the lipid nanoparticle comprises about 0.5 mol% to about 1 .5 mol% of sialic acid lipid and about 1.5 mol% to about 2.5 mol% of PEG lipid.
108. The composition of any one of the previous claims, wherein the lipid nanoparticle comprises about 1 mol% sialic acid lipid and about 2 mol% PEG lipid.
109. The composition of any one of the previous claims, wherein the lipid nanoparticle comprises about 45 mol % to about 55 mol % ionizable lipid. no. The composition of any one of the previous claims, wherein the lipid nanoparticle comprises about 35 mol % to about 45 mol % structural lipid.
111. The composition of any one of the previous claims, wherein the lipid nanoparticle comprises about 8 mol % to about 12 mol % phospholipid.
112. The composition of any one of the previous claims, wherein the ionizable lipid is compound 1-18, 1-25, 1-301, II-6, or VI-4.
113. The composition of any one of the previous claims, wherein the phospholipid is DMPS, DSPC, DOPE, DOPC, POPE, or POPC.
114. The composition of any one of the previous claims, wherein the structural lipid is cholesterol .
115. The composition of any one of the previous claims, wherein the PEG lipid is PEG- DMG or PL-02.
116. The composition of any one of the previous claims, wherein the sialic acid lipid is compound 1 or compound 9.
117. The composition of any one of the previous claims, wherein the sialic acid lipid is compound 1.
118. The composition of any one of the previous claims, wherein: the ionizable lipid is 1-18; the PEG lipid is PL-02; the structural lipid is cholesterol; the phospholipid is DSPC; and the sialic acid lipid is compound 1 .
119. The composition of any one of the previous claims, wherein the target nucleic acid, comprises a sequence of CD33.
120. The composition of any one of the previous claims, wherein the CRISPR nuclease comprises Cas9.
121. A method of editing a sequence of a target nucleic acid in a cell, comprising contacting the cell with the composition of any one of the previous claims.
122. The method of claim 121. wherein the cell comprises a hematopoietic stem and progenitor cell (HSPC), an erythroid progenitor cell (EPC), a myeloid cell, and/or a lymphoid cell in a subject.
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