Lipid Composition for In Vivo Delivery

Abstract

The present specification provides compositions, methods, and kits for inducing an immune response in a subject. In an aspect, a lipid composition is described that includes at least one ionizable lipid containing a charge (N), at least one peptide, and a nucleic acid molecule containing a charge (P). In an aspect, a method for delivery of a payload to immune cells is provided that uses a lipid composition including at least one ionizable lipid, at least one endosome-releasing peptide, and a payload. TIFF2025524562000039.tif140144

Description

Technical Field

[0001] (Related Applications) This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 510,733, filed Jun. 28, 2023, and U.S. Provisional Patent Application No. 63 / 357,584, filed Jun. 30, 2022, the contents of which are hereby incorporated by reference in their entirety as if fully set forth herein.

[0002] (Sequence Listing) This application includes a Sequence Listing submitted electronically in ST.26 XML format, which is hereby incorporated by reference in its entirety. The XML document created on Jun. 28, 2023, is named TP109375WO1_SL.XML and is 34,000 bytes in size.

[0003] (Field of the Invention) The present invention is in the field of lipid compositions and formulations suitable for the delivery of one or more bioactive agents to cells (e.g., immune cells), and methods and kits for using the same.

Background Art

[0004] It remains a challenge to design lipid compositions and formulations that can target a payload to a specific organ, tissue, or cell type without using standard biomolecular targeting techniques. Improved compositions and methods for delivering bioactive payloads to specific tissues and / or cell types, particularly immune system tissues and cells, are highly desired. Compositions and methods are provided herein to address this and other needs in the art.

Summary of the Invention

[0005] In one aspect, a method for delivering a payload to splenocytes in a subject, the method comprising: (i) providing a lipid complex comprising at least one ionizable lipid, at least one peptide comprising the peptide LLELLESL (SEQ ID NO: 1), and at least one payload; and (ii) administering the lipid complex to the subject. In some embodiments, the lipid complex further comprises at least one neutral lipid.

[0006] In some embodiments, the peptide has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24. In other embodiments, the lipid complex further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0007] In some embodiments, the peptide is at a concentration of about 0.001 to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of about 0.001 to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of about 0.001 mg / mL. In other embodiments, the peptide is at a concentration of about 0.05 mg / mL. In other embodiments, the peptide is at a concentration of about 0.5 mg / mL.

[0008] In certain embodiments, the payload is a nucleic acid. In some embodiments, the at least one ionizable lipid comprises a charge (N), the payload nucleic acid comprises a charge (P), and the lipid complex comprises an N / P ratio of 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.0.

[0009] In some embodiments, the N / P ratio is from about 0.01 to about 0.2. In some embodiments, the N / P ratio is from about 0.05 to about 0.5. In some embodiments, the N / P ratio is from about 0.1 to about 1.0. In some embodiments, the N / P ratio is from about 0.5 to about 2.0. In some embodiments, the N / P ratio is from about 1.0 to about 5.0. In some embodiments, the N / P ratio is less than about 0.1. In some embodiments, the N / P ratio is about 0.1. In some embodiments, the N / P ratio is about 0.2. In some embodiments, the N / P ratio is about 0.5. In some embodiments, the N / P ratio is about 1.0. In some embodiments, the N / P ratio is about 2.0.

[0010] In some embodiments, the payload comprises an RNA molecule. In some embodiments, the RNA molecule comprises an mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or a combination thereof. In some embodiments, the RNA molecule comprises more than one mRNA molecule. In some embodiments, the RNA molecule comprises at least two mRNA molecules. In some embodiments, the RNA molecule comprises an sgRNA molecule and an mRNA molecule. In some embodiments, the RNA molecule comprises an sgRNA molecule.

[0011] In other embodiments, the payload further comprises a protein.

[0012] In some embodiments, the nucleic acid payload encodes an immunogen. In some embodiments, the nucleic acid payload encodes hemagglutinin (HA) or ovalbumin.

[0013] In some embodiments, the ionizable lipid comprises a lipid according to formula (I), formula (II), formula (III), formula (IV), or formula (V), or a combination thereof. In some embodiments, the ionizable lipid comprises at least one lipid according to formula (I). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (II). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (III). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (IV). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (V).

[0014] In some embodiments, the ionizable lipid comprises a lipid according to formula (IA) or formula (IB), or a combination thereof. In some embodiments, the ionizable lipid comprises a lipid according to formula (IA). In some embodiments, the ionizable lipid comprises a lipid according to formula (IB). In some embodiments, at least one ionizable lipid comprises a lipid according to formula (IA) and an ionizable lipid according to formula (IB).

[0015] In some embodiments, the ionizable lipid comprises a lipid according to formula (IIA), formula (IIB), or formula (IIC), or a combination thereof. In some embodiments, the ionizable lipid comprises a lipid according to formula (IIA). In some embodiments, the ionizable lipid comprises a lipid according to formula (IIB). In some embodiments, the ionizable lipid comprises a lipid according to formula (IIC).

[0016] In some embodiments, the neutral lipids are cholesterol, sterol, dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), lysophosphatidylethanolamine (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), lysophosphatidylcholine (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,It contains 2-dioleoyl-sn-glycero-3-phophoethanolamine (1,2-dioleoyl-sn-glycero-3-phophoethanolamine, trans DOPE), or a combination thereof.,

[0017] In other embodiments, the lipid complex comprises liposomes. In some embodiments, the lipid complex comprises lipid nanoparticles. In some embodiments, the lipid complex comprises a population of lipid nanoparticles, and the nanoparticles have a diameter of about 20 nm to about 1 μm. In some embodiments, the nanoparticles have a diameter of about 10 nm to about 900 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 800 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 700 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 6 hundred nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 500 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 400 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 300 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 100 nm. In some embodiments, the nanoparticles have a diameter of about 20 nm to about 50 nm.

[0018] In embodiments, the step of administration comprises systemic administration or local administration. In some embodiments, local administration comprises intramuscular administration or subcutaneous administration. In embodiments, the step of administration comprises intravenous administration. In embodiments, the step of administration comprises administration to the brain, spinal cord, eye, or lymph nodes of the subject.

[0019] In some embodiments, the subject includes a mammalian subject. In some embodiments, the mammal is a human.

[0020] It should be noted that in the translation of the diameter range in ID=4, "hundred" in "six hundred nm" is retained as it is in the original text for the sake of strict translation following the rules. If this is an incorrect expression in the original, it may need to be adjusted according to the actual situation.In some embodiments, the payload is delivered to dendritic cells of the spleen. In some embodiments, the lipid complex targets splenic cells of a subject by about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold or more compared to targeting by administration of a lipid complex that does not contain a peptide and contains an ionizable lipid and a payload.

[0021] Provided herein is a method for expressing a protein in splenic tissue in a subject, the method comprising administering to the subject a lipid complex described herein via systemic administration.

[0022] In an aspect, provided herein is a composition for delivery of a payload to splenic cells. Provided herein is a composition having at least one ionizable lipid, at least one peptide, wherein the peptide has and contains LLELLESL (SEQ ID NO: 1) and is present at a concentration of less than 1.0 mg / ml, and at least one payload molecule. In other embodiments, the peptide is at a concentration of about 1.0 mg / mL. In other embodiments, the peptide is at a concentration of about 0.05 mg / mL.

[0023] Also provided herein is a composition comprising at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide has and contains LLELLESL (SEQ ID NO: 1), and at least one payload comprising a nucleic acid having a charge (P). The composition has an N / P ratio of 0.01-0.2, or 0.05-0.5, or 0.1-1.0, or 0.5-2.0, or 1.0-5.0. In some embodiments, the N / P ratio is the ratio of the positively charged group (including the amine (N) group) to the negatively charged group including the phosphate (P) group and the peptide group.

[0024] Also provided herein is a composition comprising at least one ionizable lipid having a charge (N), at least one endosome-releasing peptide, and at least one payload comprising a nucleic acid having a charge (P), wherein the composition has an N / P ratio of from about 0.01 to about 0.5.

[0025] In some embodiments, the composition further comprises at least one neutral lipid. In some embodiments of the composition, the peptide has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24. In other embodiments, the composition further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0026] In some embodiments, the peptide is at a concentration of from about 0.001 to about 0.5 mg / mL, or from about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of from about 0.001 to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of from about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the peptide is at a concentration of about 0.001 mg / mL. In other embodiments, the peptide is at a concentration of about 0.05 mg / mL. In other embodiments, the peptide is at a concentration of about 0.5 mg / mL.

[0027] In some embodiments, the N / P ratio is from about 0.01 to about 0.2. In some embodiments, the N / P ratio is from about 0.05 to about 0.5. In some embodiments, the N / P ratio is from about 0.1 to about 1.0. In some embodiments, the N / P ratio is from about 0.5 to about 2.0. In some embodiments, the N / P ratio is from about 1.0 to about 5.0. In some embodiments, the N / P ratio is less than about 0.1. In some embodiments, the N / P ratio is about 0.1. In some embodiments, the N / P ratio is about 0.2. In some embodiments, the N / P ratio is about 0.5. In some embodiments, the N / P ratio is about 1.0. In some embodiments, the N / P ratio is about 2.0.

[0028] In some embodiments, the payload comprises an RNA molecule. In some embodiments, the RNA molecule comprises an mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or a combination thereof. In some embodiments, the RNA molecule comprises more than one mRNA molecule. In some embodiments, the RNA molecule comprises at least two mRNA molecules. In some embodiments, the RNA molecule comprises an sgRNA molecule and an mRNA molecule. In some embodiments, the RNA molecule comprises an sgRNA molecule.

[0029] In other embodiments, the payload further comprises a protein.

[0030] In some embodiments, the nucleic acid payload encodes an immunogen. In some embodiments, the nucleic acid payload encodes hemagglutinin (HA) or ovalbumin.

[0031] In some embodiments, the ionizable lipid comprises a lipid according to formula (I), formula (II), formula (III), formula (IV), or formula (V), or a combination thereof. In some embodiments, the ionizable lipid comprises at least one lipid according to formula (I). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (II). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (III). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (IV). In some embodiments, the ionizable lipid comprises at least one lipid according to formula (V).

[0032] In some embodiments, the ionizable lipid comprises a lipid according to formula (IA) or formula (IB), or a combination thereof. In some embodiments, the ionizable lipid comprises a lipid according to formula (IA). In some embodiments, the ionizable lipid comprises a lipid according to formula (IB). In some embodiments, at least one ionizable lipid comprises a lipid according to formula (IA) and an ionizable lipid according to formula (IB).

[0033] In some embodiments, the ionizable lipid comprises a lipid according to formula (IIA), formula (IIB), or formula (IIC), or a combination thereof. In some embodiments, the ionizable lipid comprises a lipid according to formula (IIA). In some embodiments, the ionizable lipid comprises a lipid according to formula (IIB). In some embodiments, the ionizable lipid comprises a lipid according to formula (IIC).

[0034] In some embodiments, the neutral lipid comprises cholesterol, sterol, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPhPE), lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), or a combination thereof.

[0035] In other embodiments, the composition comprises liposomes. In some embodiments, the composition comprises lipid nanoparticles. In some embodiments, the composition comprises a population of lipid nanoparticles, wherein the nanoparticles have a diameter of from about 20 nm to about 1 μm. In some embodiments, the nanoparticles have a diameter of from about 10 nm to about 900 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 800 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 700 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 600 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 500 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 400 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 300 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 100 nm. In some embodiments, the nanoparticles have a diameter of from about 20 nm to about 50 nm.

[0036] In some embodiments, the composition is for administration to a subject via intramuscular administration, subcutaneous administration, intravitreal administration, administration to the brain, or administration to the spinal cord.

[0037] In embodiments, provided herein is a method of inducing an immune response in a subject, comprising administering to the subject a composition described herein, wherein the payload is an immunogen or encodes an immunogen. In embodiments, the administering step comprises systemic or local administration, and local administration comprises intramuscular or subcutaneous administration. In embodiments, the administering step comprises intravenous administration. In embodiments, the administering step comprises administration to the subject's brain, spinal cord, eye, or lymph nodes. In some embodiments, the subject comprises a mammalian subject. In some embodiments, the mammalian is a human.

[0038] In some embodiments, methods for delivering a payload to target immune cells are provided herein, the methods including administering to a subject any of the compositions described herein via intravenous administration or via intramuscular administration.

[0039] In some embodiments, methods for targeting a payload to target immune cells are provided herein, the methods including administering to a subject a composition described herein. In embodiments, the immune cells include T cells, B cells, dendritic cells (DCs), T helper cells, cytotoxic T cells (CTLs), natural killer cells (NKs), macrophages, or combinations thereof. In other embodiments, the immune cells include splenic immune cells. In some embodiments, the immune cells include splenic dendritic cells.

[0040] In some embodiments, the composition targets the subject's immune cells about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold or more compared to targeting by administration of a composition containing an ionizable lipid and a payload that does not contain a peptide.

[0041] In an aspect, a method for preparing a population of lipid formulations containing a payload molecule, the method comprising: (a) mixing the payload molecule with a peptide, the peptide comprising LLELLESL (SEQ ID NO: 1), in an aqueous solution; and (b) injecting a lipid solution containing an ionizable lipid into the aqueous solution, the injecting comprising extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (c) generating a population of lipid formulations complexed with the payload molecule.

[0042] Also provided herein is a method for preparing a population of lipid formulations containing a payload molecule, comprising: (a) contacting a peptide comprising LLELLESL (SEQ ID NO: 1) with a lipid phase comprising an ionizable lipid; (b) contacting the components of step (a) with a payload in an aqueous solution; (c) mixing the components of step (b) by extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (d) generating a population of lipid formulations complexed with the payload molecule.

[0043] In some embodiments, the lipid solution of (b) or the lipid phase of (a) further comprises at least one neutral lipid. In some embodiments, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the payload molecule is a nucleic acid.

[0044] In another aspect, provided herein is a kit comprising a composition having at least one ionizable lipid and at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 1). In some embodiments, the kit further comprises at least one neutral lipid. In some embodiments, the ionizable lipid and the neutral lipid are in a separate container from the peptide. In some embodiments, the ionizable lipid and the neutral lipid are in the same container as the peptide.

[0045] Other aspects of the invention are disclosed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0046]

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Modes for Carrying Out the Invention

[0047] The present inventors have developed a composition that is particularly effective for in vivo delivery of payload molecules to specific tissues and / or cell types, particularly immune system tissues and cells. The composition contains at least one ionizable lipid, an endosome-releasing peptide, and a payload, and specifically provides effective and efficient delivery of the payload to the spleen, particularly dendritic cells in the spleen, after intravenous administration. After local administration (e.g., intramuscular injection), the composition specifically provides effective and efficient delivery of the payload to the injection site tissue and local immune cells. For example, antigen-specific humoral and cellular immune responses were obtained after systemic or local administration of mRNA encoding an antigen complexed with the provided formulation.

[0048] In therapeutic applications, for example, it is important to achieve the desired biological endpoint while administering the minimum amount of formulation in order to reduce or lower the level of associated toxicity and / or off-target effects. As demonstrated herein, the use of peptides in combination with ionizable lipids in the provided compositions results in effective and efficient delivery / uptake of the payload nucleic acid even at low levels of lipids and / or nucleic acids in the composition. This improved efficiency allows for the administration of fewer formulations (e.g., lower mRNA and / or lipid levels) to achieve a therapeutic effect, such as a specific immune response.

[0049] Exemplary payloads for delivery to immune system tissues and cells via the provided lipid complex compositions include nucleic acid molecules, protein molecules, and / or other bioactive agents. In some embodiments, the payload for delivery to immune cells is a therapeutic or diagnostic agent. In some embodiments, the lipid complex composition further comprises at least one neutral lipid.

[0050] Provided herein are compositions, methods, and kits for inducing an immune response in a subject. In an aspect, a lipid composition is described that comprises at least one ionizable lipid comprising a charge (N), at least one peptide comprising the sequence LLELLESL (SEQ ID NO: 1), and a nucleic acid molecule comprising a charge (P), the composition comprising an N / P ratio of 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.0. In some embodiments, the N / P ratio can be less than about 0.1. In some embodiments, the N / P ratio is the ratio of the positively charged group (including the amine (N) group) to the negatively charged group comprising the phosphate (P) group and the peptide group. In some embodiments, the lipid composition further comprises at least one neutral lipid. In some embodiments, the peptide comprises SEQ ID NO: 1 and a polycationic nucleic acid binding domain. In some embodiments, the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

[0051] In another aspect, a composition for delivery of a payload to splenocytes is provided, the composition comprising at least one ionizable lipid and at least one peptide comprising the sequence LLELLESL (SEQ ID NO: 1). In some embodiments, the composition for delivery of a payload to splenocytes further comprises at least one neutral lipid. In some embodiments, in the composition for delivery of a payload to splenocytes, the peptide comprising SEQ ID NO: 1 is present at a concentration of less than 1.0 mg / ml. In some embodiments, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the composition for delivery of a payload to splenocytes further comprises at least one payload.

[0052] Provided herein are, inter alia, methods, compositions, and kits for targeting a payload to immune cells, such as splenocytes, in vitro, ex vivo, or in a subject. In some embodiments of such methods,

[0053] Provided herein are, inter alia, methods, compositions, and kits for inducing an immune response in a subject.

[0054] Provided herein are, inter alia, methods and compositions for generating a population of lipid formulations containing a payload.

[0055] General Definitions The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended claims. Abbreviations used herein have their conventional meanings within the technical fields of chemistry and biology.

[0056] Although various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0057] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and theses, are hereby expressly incorporated by reference in their entirety for any purpose.

[0058] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994), and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0059] The term "about" when used in connection with a numerical range, cutoff, or substantial value is used to indicate that the recited value can vary by up to 25% from the recited value. Since many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can vary between different experiments and will often fluctuate. The values used herein should not be regarded as being overly limiting by virtue of this inherent variability. The term "about" is used to encompass variations of ±25% or less, ±20% or less, 10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term "about".

[0060] In the description and claims of this specification, phrases such as "at least one" or "one or more of" may appear, followed by a list of elements or features. The term "and / or" may also appear in a list of two or more elements or features. Unless the context in which such terms are used is implicitly or explicitly contradictory, such phrases are intended to mean any of the recited elements or features individually, or any of the recited elements or features in combination with any of the other recited elements or features. For example, "at least one of A and B", "one or more of A and B", and "A and / or B" are each intended to mean "only A, only B, or A and B together". A similar interpretation is also intended for lists containing three or more items. For example, "at least one of A, B, and C", "one or more of A, B, and C", and "A, B, and / or C" are each intended to mean "only A, only B, only C, A and B together, A and C together, B and C together, or A and B and C together". In addition, the use of the term "based on" in the above and in the claims is intended to mean "based at least in part on" so as to allow features or elements not recited.

[0061] When a parameter range is provided, it is understood that all integers within that range and tenths thereof are also provided by the present invention. For example, "0.2 to 5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc., up to and including 5.0 mg.

[0062] Compounds are generally described herein using standard nomenclature. For enumerated compounds having asymmetric centers, all stereoisomers of the compound and mixtures thereof are included unless otherwise specified. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. If an enumerated compound exists in various tautomeric forms, the compound is intended to include all tautomeric forms. Certain compounds are described herein using general formulas that include variables (e.g., X, L1, L2, L3, Y, etc.). Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that appears more than once in the formula is defined independently at each occurrence. When a moiety is described as being “independently” selected from a group, each moiety is selected independently of one another. Thus, each moiety can be the same as or different from one or more other moieties. The number of carbon atoms in a hydrocarbyl moiety can be indicated by the prefix “C x ~C y ”, where x is the minimum number of carbon atoms in the moiety and y is the maximum number. Thus, for example, “C1~C6 alkyl” refers to an alkyl substituent containing from 1 to 6 carbon atoms. As a further illustration, C3~C6 cycloalkyl means a saturated hydrocarbyl ring containing from 3 to 6 carbon ring atoms. A prefix attached to a multi-component substituent applies only to the first component following immediately after the prefix. By way of illustration, the term “carbocyclic alkyl” contains two components, namely, carbocyclic and alkyl. Thus, for example, C3~C6 carbocyclic C1~C6 alkyl refers to a C3~C6 carbocyclic attached through a C1~C6 alkyl group to the parent molecular moiety.

[0063] Unless otherwise specified, when a linking element connects two other elements in the depicted chemical structure, the left-most described component of the linking element is bonded to the left element in the depicted structure, and the right-most described component of the linking element is bonded to the right element in the depicted structure. By way of illustration, if the chemical structure is -L S -M-L S ”- and M is -N(RB ) When it is -S(O)-, the chemical structure is -L S -N(R B )-S(O)-L S ”- is.

[0064] When the connecting element in the described structure is a bond, the element on the left side of the connecting element is directly bonded to the element on the right side of the connecting element through a covalent bond. For example, if the chemical structure is -L S -M-L S ’ is described and M is selected as a bond, the chemical structure is -L S -L S ”- becomes. When two or more adjacent connecting elements in the described structure are bonds, the element on the left side of these connecting elements is directly bonded to the element on the right side of these connecting elements through a covalent bond. For example, if the chemical structure is -L S -M-L S ”-M’-L S ”- is described and M and L S ’ are selected as bonds, the chemical structure is -L S -M’-L S ”- becomes. Similarly, if the chemical structure is -L S -M-L S ”-M’-L S ”- is described and M, L S ’ and M’ are bonds, the chemical structure is -L S -L S ” becomes. When a chemical formula is used to describe a part, the dash indicates a part of the moiety with a free valence.

[0065] When a moiety is described as being "optionally substituted", the moiety can be either substituted or unsubstituted. When a moiety is described as being optionally substituted with up to a specific number of non-hydrogen radicals, the moiety can be unsubstituted or substituted with up to the lesser of the specific number of non-hydrogen radicals or the maximum number of substitutable positions on that moiety. Thus, for example, when a moiety is described as a heterocyclic ring optionally substituted with up to 3 non-hydrogen radicals, any heterocyclic ring having less than 3 substitutable positions is optionally substituted only with up to the same number of non-hydrogen radicals as the heterocyclic ring has substitutable positions. For example, tetrazolyl (which has only 1 substitutable position) is optionally substituted with up to 1 non-hydrogen radical. Similarly, when amino nitrogen is described as being optionally substituted with up to 2 non-hydrogen radicals, primary amino nitrogen is optionally substituted with up to 2 non-hydrogen radicals, while secondary amino nitrogen is optionally substituted only with up to 1 non-hydrogen radical.

[0066] When a moiety is substituted with oxo or thioxo, it means that the moiety contains a carbon atom covalently bonded to at least 2 hydrogens (e.g., CH2) and the 2 hydrogen radicals are substituted with oxo or thioxo to form C=O or C=S, respectively.

[0067] The term "alkenyl" means a straight or branched hydrocarbyl chain containing one or more double bonds. Each carbon-carbon double bond can have either E (cis) or Z (trans) geometry within the alkenyl moiety with respect to the groups substituted on the double bond carbons. Examples of alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E-, and Z,Z-hexadienyl.

[0068] The term "alkenylene" refers to a divalent unsaturated hydrocarbyl chain which may be straight-chain or branched-chain and has at least one carbon-carbon double bond. Non-limiting examples of alkenylene groups include -C(H)=C(H)-, -C(H)=C(H)-CH2-, -C(H)=C(H)-CH2-CH2-, -CH2-C(H)=C(H)-CH2-, -C(H)=C(H)-CH-(CH3)-, and -CH2-C(H)=C(H)-CH-(CH2CH3)-.

[0069] The term "alkyl" means a straight-chain or branched saturated hydrocarbyl chain. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, iso-amyl, and hexyl.

[0070] The term "alkylene" denotes a divalent saturated hydrocarbyl chain which may be straight-chain or branched-chain. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-.

[0071] The term "alkynyl" means a straight-chain or branched hydrocarbyl chain containing one or more triple bonds. Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 3-propynyl, decynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

[0072] The term "alkynyl", alone or in combination with any other term, refers to a straight-chain or branched-chain hydrocarbon radical having one or more triple bonds and containing a specified number of carbon atoms, or, when no number is specified, containing from 2 to about 10 carbon atoms in one embodiment. Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl, etc.

[0073] The term "alkoxy" refers to an alkyl ether radical, and the term "alkyl" is defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

[0074] The term "aryl", alone or in combination with any other term, is optionally substituted with one or more substituents selected from alkyl, alkoxy, (e.g., methoxy), nitro, halogen, (e.g., chloro), amino, carboxylate, and hydroxy, and has a specified number of carbon atoms, in one embodiment 6 to 15 carbon atoms (i.e., (C 6~15 aryl), in another embodiment 6 to 10 carbon atoms (i.e., (C 6~10 aryl), and refers to a carbocyclic aromatic radical (such as phenyl or naphthyl). Examples of aryl radicals include, but are not limited to, phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl, and the like.

[0075] The term "aralkyl", alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl, or the like.

[0076] The term "aralkoxycarbonyl", alone or in combination, means a radical of the formula -C(O)-O-aralkyl in which the term "aralkyl" has the meaning defined above. An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.

[0077] The term "aryloxy", alone or in combination, means a radical of the formula aryl-O- in which the term "aryl" has the meaning defined above.

[0078] The term "alkynylene" refers to a divalent unsaturated hydrocarbon group which can be straight-chain or branched-chain and has at least one carbon-carbon triple bond. Representative alkynylene groups include, by way of example, -C≡C-, -C≡C-CH2-, -C≡C-CH2-CH2-, -CH2-C≡C-CH2-, -C≡C-CH(CH3)-, and -CH2-C≡C-CH(CH2CH3)-.

[0079] The term "alkanoyl", alone or in combination, means an acyl radical derived from an alkane carboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, and the like.

[0080] The term "aryloxyalkanoyl" means an acyl radical of the formula aryl-O-alkanoyl, wherein aryl and alkanoyl have the meanings given above.

[0081] The term "aralkanoyl" means an acyl radical derived from an aryl-substituted alkane carboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, and the like.

[0082] The term "aroyl" means an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, optionally substituted benzoic acids or naphthoic acids, for example, benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4-benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3-(benzyloxyformamide)-2-naphthoyl, and the like.

[0083] The term "aminocarbonyl" alone or in combination means an aminocarbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid, where the amino group can be a primary, secondary, or tertiary amino group that continues a substituent selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals, etc.

[0084] The term "aminoalkanoyl" means an acyl radical derived from an amino-substituted alkane carboxylic acid, where the amino group can be a primary, secondary, or tertiary amino group that includes a substituent selected from the group consisting of hydrogen, cycloalkyl, cycloalkylalkyl radicals, etc., examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.

[0085] The terms "carbocyclic", "carbocyclic ring", or "carbocyclyl" refer to a saturated (e.g., "cycloalkyl"), partially saturated (e.g., "cycloalkenyl" or "cycloalkynyl"), or fully unsaturated (e.g., "aryl") 3- to 8-membered carbocyclic ring system containing 0 heteroatom ring atoms. "Ring atom" or "ring member" is an atom that is joined together to form one or more rings. Carbocyclyl can be, but is not limited to, a monocyclic, two-fused ring, or a bridged or spiro ring. Substituted carbocyclyl can have either a cis or trans configuration. Representative examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, decalinyl, and norpinanyl. The carbocyclic group can be attached to the parent molecular moiety through any substitutable carbocyclic atom. When the carbocyclic group is a divalent moiety that links two other elements in the depicted chemical structure, the carbocyclic group can be attached to the two other elements through any two substitutable ring atoms. Similarly, when the carbocyclic group is a trivalent moiety that links three other elements in the depicted chemical structure, the carbocyclic group can be attached to the three other elements through any three substitutable ring atoms, respectively. The carbocyclic ring can be joined by any in-ring carbon atom that results in a stable structure. The carbocyclic ring in one embodiment has 5 to 7 carbons.

[0086] The term "cycloalkyl" refers to a saturated carbocyclyl group containing 0 heteroatom ring members. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl, and norpinanyl.

[0087] The term "cycloalkyl", alone or in combination, means an alkyl radical that contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0088] The term "cycloalkylalkyl" means an alkyl radical as defined above that is substituted by a cycloalkyl radical containing from about 3 to about 8 carbon atoms, and in one embodiment from about 3 to about 6 carbon atoms.

[0089] The term "cycloalkylcarbonyl" means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, or a benzofused monocyclic cycloalkanecarboxylic acid optionally substituted by, for example, alkanoylamino such as 1,2,3,4-tetrahydro-2-naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0090] The term "cycloalkylalkoxycarbonyl" means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl - O - COOH, where cycloalkylalkyl has the meaning given above.

[0091] The term "carboscyclic alkyl" refers to a carbocyclic group attached to the parent molecule moiety through an alkylene group. For example, C3 - C6 carbocyclic C1 - C6 alkyl refers to a C3 - C6 carbocyclic group attached to the parent molecule moiety through a C1 - C6 alkylene.

[0092] The term "cycloalkenyl" refers to a non - aromatic partially unsaturated carbocyclic moiety having 0 heteroatom ring members. Representative examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, and octahydronaphthalenyl.

[0093] The prefix "halo" indicates that the substituent to which the prefix is attached is substituted with one or more independently selected halogen radicals. For example, "C1-C6 haloalkyl" means a C1-C6 alkyl substituent in which one or more hydrogen atoms are replaced with independently selected halogen radicals. Non-limiting examples of C1-C6 haloalkyl include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and 1,1,1-trifluoroethyl. It should be recognized that when a substituent is substituted by more than one halogen radical, those halogen radicals may be the same or different (unless otherwise specified).

[0094] The terms "heterocyclic", "heterocyclo", or "heterocyclyl" refer to a saturated (e.g., "heterocycloalkyl"), partially unsaturated (e.g., "heterocycloalkenyl" or "heterocycloalkynyl"), or fully unsaturated (e.g., "heteroaryl") ring system in which at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen, or sulfur) and the remaining ring atoms are independently selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. The heterocyclic ring can be, but is not limited to, a monocyclic, two-fused ring, or a bridged or spiro ring. The heterocyclic group can be linked to the parent molecular moiety through any replaceable carbon or nitrogen atom in the group. When the heterocyclic group is a divalent moiety that links two other elements in the depicted chemical structure, the heterocyclic group can be bonded to the two other elements through any two replaceable ring atoms. Similarly, when the heterocyclic group is a trivalent moiety that links three other elements in the depicted chemical structure, the heterocyclic group can be bonded to the three other elements through any three replaceable ring atoms, respectively.

[0095] In this compound, "Het" represents a heterocyclic ring containing 4 to 12 carbon atoms in which at least one nitrogen atom is present in the ring. The heterocyclyl can be, but is not limited to, a monocyclic ring containing a single ring.Non-limiting examples of monocyclic rings include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiadiazolyl, oxathiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl (also known as "azoxymil"), 1,2,5-oxadiazolyl (also known as "furazanyl"), and 1,3,4-oxadiazolyl), oxatriazolyl (including 1,2,3,4-oxatriazolyl and 1,2,3,5-oxatriazolyl), dioxazolyl (including 1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl, and 1,3,4-dioxazolyl), pyridinyl, piperidinyl, diazinyl (pyridazylidene also known as "1,2-diazinyl"), pyrimidinyl (also known as "1,3-diazinyl"), and pyrazinyl (also known as "1,4-diazinyl"), piperazinyl, triazinyl (including s-triazinyl (also known as "1,3,5-triazinyl"), as-triazinyl (also known as "1,2,4-triazinyl"), and v-triazinyl (also known as "1,2,3-triazinyl")), oxazinyl (including 1,2,3-oxazinyl, 1,3,2-oxazinyl, 1,3,6-oxazinyl (also known as "pentoxazolyl"), 1,2,6-oxazinyl, and 1,4-oxazinyl), isoxazinyl (including o-isoxazinyl and p-isoxazinyl), oxazolidinyl, isoxazolidinyl, oxathiadinyl (including 1,2,5-oxathiadinyl or 1,2,6-oxathiadinyl), oxadiazinyl (including 1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, azepinyl, and diazepinyl.

[0096] Heterocyclyl can also be, but is not limited to, for example, naphthyridinyl (including [1,8]naphthyridinyl and [1,6]naphthyridinyl), thiazolopyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyrindinyl, pyranopyrrolyl, 4H - quinolidinyl, purinyl, pyridopyridinyl (including pyrido[3,4 - b] - pyridinyl, pyrido[3,2 - b] - pyridinyl, and pyrido[4,3 - b] - pyridinyl), pyridopyrimidine, and pteridinyl, etc., which can be bicyclic containing two fused rings. Other non - limiting examples of fused - ring heterocycles include indolyl, isoindolyl, indoleninyl (also known as "pseudoindolyl"), isoindazolyl (also known as "benzpyrazolyl" or indazolyl), benzazinyl (including quinolinyl (also known as "1 - benzazinyl") and isoquinolinyl (also known as "2 - benzazinyl")), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including cinnolinyl (also known as "1,2 - benzodiazinyl") and quinazolinyl (also known as "1,3 - benzodiazinyl")), benzothiazolyl, 4,5,6,7 - tetrahydrobenzothiazolyl, benzothiadiazolyl, benzimidazolyl, benzotriazolyl, benzoxazinyl (including 1,3,2 - benzoxazinyl, 1,4,2 - benzoxazinyl, 2,3,1 - benzoxazinyl, and 3,1,4 - benzoxazinyl), benzisoxazinyl (including 1,2 - benzisoxazinyl and 1,4 - benzisoxazinyl), and tetrahydroisoquinolinyl, etc., benzofused heterocyclyl.

[0097] Heterocyclyl can also be, but is not limited to, for example, a spiro ring system such as 1,4-dioxa-8-azaspiro[4.5]decan-1-yl. Heterocyclyl can contain one or more sulfur atoms as ring members, and in some cases, the sulfur atoms are oxidized to SO or SO2. The nitrogen heteroatoms in heterocyclyl may or may not be quaternized, and may or may not be oxidized to N-oxides. In addition, the nitrogen heteroatoms may or may not be N-protected.

[0098] The heterocyclic or carbocyclic ring can be further substituted. Unless otherwise specified, the term "substituted" means that one, two, or more than three of the hydrogen atoms are replaced with -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO2, -N3, -CN, -NH2, protected amino, oxo, thioxo, -NH-C2-C8-alkenyl, -NH-C2-C8-alkynyl, -NH-C3-C 12 -cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C1-C 12 -alkyl, -O-C2-C8-alkenyl, alkynyl, -O-C3-C 12 -cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(O)-C1-C 12 -alkyl, -C(O)-C2-C8-alkenyl, -C(O)-C2-C8-alkynyl, -C(O)-C3-C 12 -cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C 12 -alkyl, -CONH-C2-C8-alkenyl, -CONH-C2-C8-alkynyl, -CONH-C3-C 12 -cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1-C 12 -alkyl, -OCO2-C2-C8-alkenyl, -OCO2-C2-C8-alkynyl, -OCO2-C3-C 12-Cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2 -OCONH-C1~C 12 -alkyl, -OCONH-C2~C8-alkenyl, -OCONH-C2~C8-alkynyl, -OCONH-C3~C 12 Cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC(O)-C1~C 12 -alkyl, -NHC(O)-C2~C8-alkenyl, -NHC(O)-C2~C8-alkynyl, -NHC(O)-C3~C 12 -Cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-C1~C 12 -alkyl, -NHCO2-C2~C8-alkenyl, -NHCO2-C2~C8-alkynyl, -NHCO2-C3~C 12 -Cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH-C1~C 12 -alkyl, -NHC(O)NH-C2~C8-alkenyl, -NHC(O)NH-C2~C8-alkynyl, -NHC(O)NH-C3~C 12 -Cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1~C 12 -alkyl, -NHC(S)NH-C2~C8-alkenyl, -NHC(S)NH-C2~C8-alkynyl, -NHC(S)NH-C3~C 12 -Cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2 -NHC(NH)NH-C1~C 12 -alkyl, -NHC(NH)NH-C2~C8-alkenyl, NHC(NH)NH-C2~C8-alkynyl, -NHC(NH)NH-C3~C 12-Cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C1-C 12 -alkyl, -NHC(NH)-C2-C8-alkenyl, -NHC(NH)-C2-C8-alkynyl, -NHC(NH)-C3-C 12 -Cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C 12 -alkyl, -C(NH)NH-C2-C8-alkenyl, -C(NH)NH-C2-C8-alkynyl, -C(NH)NH-C3-C 12 -Cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-C1-C 12 -alkyl, -S(O)-C2-C8-alkenyl, -S(O)-C2-C8-alkynyl, -S(O)-C3-C 12 -Cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SO2NH2-SO2NH-C1-C 12 -alkyl, -SO2NH-C2-C8-alkenyl, -SO2NH-C2-C8-alkynyl, -SO2NH-C3-C 12 -Cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1-C 12 -alkyl, -NHSO2-C2-C8-alkenyl, -NHSO2-C2-C8-alkynyl, -NHSO2-C3-C 12 -Cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2-CH2SO2CH3, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, -C3-C 12 -Cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-C1-C 12-alkyl, -S-C2-C8-alkenyl, -S-C2-C8-alkynyl, -S-C3-C 12 Refers to substitution by independently replacing with substituents including, but not limited to, -cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It is understood that aryl, heteroaryl, alkyl, etc. may be further substituted.

[0099] The term "N-protecting group" or "N-protected" refers to a group capable of protecting an amino group from unwanted reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3 rdIt is described in (ed., John Wiley & Sons, NY (1999)). Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, or 4-nitrobenzoyl, sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl, sulfenyl groups such as phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl (trityl-S-), sulfinyl groups such as p-methylphenylsulfinyl (p-methylphenyl-S(O)-) or t-butylsulfinyl (t-Bu-S(O)-), carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyloxy, phenoxycarbonyl, 4-nitrophenoxycarbonyloxy, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, alkyl groups such as benzyl, p-methoxybenzyl, triphenylmethyl, or benzyloxymethyl, p-methoxyphenyl, and silyl groups such as trimethylsilyl.Preferred N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0100] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0101] The terms "biologically active agent", "bioactive agent", etc. generally refer to a composition, complex, compound, or molecule that has a biological effect, or modifies, causes, promotes, enhances, blocks, or reduces a biological effect, or enhances or limits the production or activity of a second molecule having a biological effect, reacts with, and / or binds to the second molecule. The second molecule can be an endogenous molecule (e.g., a molecule such as a protein or nucleic acid normally present in the target cell), but it need not be. Biological effects can include those that stimulate or cause an immunoreactive response, affect biological processes in cells, tissues, or organisms (e.g., in animals), confer biological processes in pathogens or parasites, generate or are caused to generate a detectable signal, regulate the expression of a protein or polypeptide, stop or inhibit the expression of a protein or polypeptide, or cause or enhance the expression of a protein or polypeptide, but are not limited thereto. Biologically active compositions, complexes, compounds, or molecules are used in investigative, therapeutic, prophylactic, and diagnostic methods and compositions and can generally act to cause.

[0102] As used herein, the term "transfection enhancer" or "transfection potentiator" refers to a compound that, when added to a transfection agent, increases the efficiency of transfection (i.e., increases the proportion of cells that are transfected), increases the level of expression of the transfection agent, or reduces the amount of payload required to elicit a biological response, such as a nucleic acid or protein, or any combination of the above enhancements. In some embodiments, the transfection enhancer also serves to deliver molecules that are useful for downregulating the expression of, for example, siRNA, LNA, etc. In some embodiments, the transfection potentiator is a peptide that includes, for example, a cell surface ligand, a fusogen, and / or a nuclear localization agent such as a nuclear receptor ligand peptide. The transfection potentiator includes, for example, the peptides and polypeptides described in Table 1 of U.S. Patent No. 10,538,784, the contents of which are incorporated herein by reference in their entirety.

[0103] As used herein, the term "nucleic acid binding moiety" refers to a compound or molecule capable of binding to a nucleic acid. In some embodiments, the binding molecule is capable of non-covalently binding to the nucleic acid, while in other embodiments, the binding molecule covalently binds to a transfection enhancer, a cell surface ligand, a nuclear localization sequence, and / or a fusogen. Examples of binding molecules include, but are not limited to, spermine, spermine derivatives, spermidine, histones or fragments thereof, protamines or fragments thereof, HMG proteins or fragments thereof, polylysine, polyarginine, polyhistidine, polyamines, and cationic peptides, nucleic acid intercalators, protein nucleic acid sequences, or aptamers. In addition, this includes, but is not limited to, analogs or derivatives of the above compounds.

[0104] As used herein, the term "polycationic nucleic acid binding moiety" refers to a moiety at physiological pH that enables a moiety containing multiple positive charges to bind to a negatively charged nucleic acid. The polycationic nucleic acid binding moiety can be linked to a transfection enhancer such as, for example, a cell surface ligand, a fusogen, and / or a nuclear localization peptide. The binding can be a covalent bond. Suitable polycationic nucleic acid binding moieties include polyamines and polybasic peptides containing, for example, multiple lysine, arginine, ornithine, or histidine residues, such as about 8 to 20 such residues. Non-limiting examples are cationic peptides that are repeats of lysine or arginine, such as sequences having 8 to 20 lysine residues or 8 to 20 arginine residues.

[0105] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, and the polymer can be conjugated to a moiety that does not consist of amino acids in embodiments. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A "fusion protein" refers to a chimeric protein that encodes two or more distinct protein sequences that are recombinantly expressed as a single moiety or chemically synthesized.

[0106] As used herein, the terms "lytic agent" or "endosome releasing agent" refer to a molecule, compound, protein, or peptide that can decompose the endosomal membrane or cell membrane and release a transfection agent such as a payload transporter containing an RNA or DNA transporter into the cytoplasm of a cell. The term includes, but is not limited to, viruses, synthetic compounds, fusion peptides, cell-penetrating peptides, lytic peptides, or derivatives thereof. The term "cell-lytic peptide" refers to a chemical group that penetrates the membrane such that the structural organization and integrity of the membrane are lost. As a result of the presence of the lytic agent, the membrane undergoes lysis, fusion, or both. Examples of lytic agents / endosome releasing agents include chloroquine, polyamines, and polyamidoamines. Suitable agents are described, for example, in Pei and Buyanova, Bioconjugate Chem, 30:273-283 (2009), and Juliano, Nucleic Acid Therapeutics, 28:166-177 (2018).

[0107] The term "surface ligand" or "cell surface ligand" refers to a chemical compound or structure that binds to a cell surface receptor. As used herein, the term "cell surface receptor" refers to a specific chemical group on the surface of a cell to which a ligand can bind. A cell surface receptor is specific for a particular cell, i.e., it can be predominantly found in one cell and not in another type of cell (e.g., the LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor promotes the internalization of the ligand and the bound molecule. Examples of cell surface receptors include, but are not limited to, folate receptor, biotin receptor, lipoic acid receptor, low density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II / cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage-derived morphogenetic protein receptor, or glycosylphosphatidylinositol (GPI) anchor type proteins (e.g., β-adrenergic receptor, T cell activation protein, Thy-1 protein, GPI anchor type 5' nucleotidase). These are non-limiting examples.

[0108] A "receptor" is a molecule to which a ligand binds specifically and with relatively high affinity. Receptors are usually proteins or glycoproteins, but can also be glycolipids, lipopolysaccharides, glycosaminoglycans, or the glycocalyx. For the purposes of the present disclosure, an epitope to which an antibody or fragment thereof binds is interpreted as a receptor because the antigen:antibody complex undergoes endocytosis. Further, surface ligands include anything that can enter a cell through cytosis (e.g., endocytosis, potocytosis, pinocytosis). As used herein, the term "ligand" refers to a chemical compound or structure that binds to a receptor. This includes ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptide, but is not limited thereto. A ligand can also be a plant virus movement protein or a peptide derived from such a protein. Suitable peptides and proteins are described, for example, in U.S. Patent No. 10,538,784, the contents of which are incorporated herein by reference in their entirety. One of ordinary skill in the art will readily recognize that the ligand selected depends on which receptor is being bound. Since different types of cells have different receptors, this provides one way to target a payload, such as a polypeptide or nucleic acid molecule, to a specific cell type depending on which cell surface ligand is used. Thus, the use of cell surface ligands can depend on the targeted cell type.

[0109] Composition Provided herein is a lipid composition comprising at least one ionizable lipid, at least one peptide comprising the sequence LLELLESL (SEQ ID NO: 1), and at least one payload molecule. In some embodiments, the lipid composition further comprises at least one neutral lipid. In some embodiments, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 1). In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-24.

[0110] Exemplary payloads for delivery to tissues and cells via the provided lipid complex compositions include nucleic acid molecules, protein molecules, and / or other bioactive agents. In some embodiments, the payload for delivery to immune cells is a therapeutic or diagnostic agent.

[0111] In some embodiments of the lipid composition, the payload, lipids, and their amounts, such as one or more nucleic acids (e.g., mRNA, siRNA, sgRNA), can be selected to provide a specific N / P ratio. The N / P ratio of the composition refers to the molar ratio of ionizable nitrogen atoms in one or more lipids to the number of phosphate groups in the nucleic acid (e.g., RNA) at physiological pH. For example, Schoenmaker et al (International Journal of Pharmaceutics; 601(2021), incorporated herein by reference in its entirety) discuss the N / P ratio of RNA-lipid nanoparticles (e.g., mRNA and siRNA payloads), for example, in Table 1 and on page 4.

[0112] In certain embodiments, a lower N / P ratio is preferred. As used herein, there is provided a lipid composition comprising at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), and a nucleic acid molecule comprising a charge (P), the composition comprising an N / P ratio of 0.01, or 0.02, or 0.04, or 0.06, or 0.08, or 0.10, or 0.12, or 0.14, or 0.16, or 0.18, or 0.20. As used herein, there is provided a lipid composition comprising at least one ionizable lipid having a charge (N), at least one neutral lipid, at least one peptide, wherein the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), and a nucleic acid molecule comprising a charge (P), the composition comprising an N / P ratio of 0.01, or 0.02, or 0.04, or 0.06, or 0.08, or 0.10, or 0.12, or 0.14, or 0.16, or 0.18, or 0.20. In other examples, the N / P ratio is from 0.01 to 0.10. In other examples, the N / P ratio is from 0.01 to 0.20. In other examples, the N / P ratio is from 0.01 to 0.25. In other examples, the N / P ratio is from 0.01 to 0.33. In other examples, the N / P ratio is from 0.01 to 0.5. In other examples, the N / P ratio is from 0.01 to 1. In other examples, the N / P ratio is from 0.05 to 0.1. In other examples, the N / P ratio is from 0.05 to 0.125. In other examples, the N / P ratio is from 0.5 to 0.15. In other examples, the N / P ratio is from 0.05 to 0.167. In other examples, the N / P ratio is from 0.05 to 0.20. In other examples, the N / P ratio is from 0.05 to 0.25. In other examples, the N / P ratio is from 0.05 to 0.33. In other examples, the N / P ratio is from 0.05 to 0.5. In other examples, the N / P ratio is from 0.05 to 1.0. In some embodiments, the N / P ratio can be less than about 0.1. In some embodiments, the N / P ratio is 0.1. In some embodiments, the N / P ratio is 0.025. In other embodiments, the N / P ratio is 0.01. In some embodiments, the N / P ratio is 0.005.

[0113] In certain embodiments, one or more nucleic acids (e.g., mRNA, siRNA, sgRNA), lipids, and their amounts can be selected to provide an N / P ratio of about 2.0 to about 8.0, such as 2, 3, 4, 5, 6, 7, and 8. In certain embodiments, the N / P ratio can be from about 2.0 to about 5.0. In some embodiments, the N / P ratio can be about 4.0. In other embodiments, the N / P ratio is from about 5 to about 8. For example, the N / P ratio can be about 5.0, about 5.5, about 5.67, about 6.0, about 6.5, or about 7.0.

[0114] In other embodiments, one or more nucleic acids, lipids, and their amounts can be selected to provide an N / P ratio of about 5 to about 50, such as 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50. In certain embodiments, the N / P ratio can be from about 5 to about 10. In other embodiments, the N / P ratio is from about 5 to about 20. In other embodiments, the N / P ratio can be from about 10 to about 20, from about 10 to about 30, from about 15 to about 30, from about 15 to about 40, from about 20 to about 30, from about 20 to about 40, from about 20 to about 50, from about 30 to about 50, from about 30 to about 40, or from about 35 to about 50.

[0115] The ionizable lipid and peptide compositions provided herein include complexes in the form of lipid nanoparticles, liposomes (e.g., lipid vesicles), and lipoplexes. As used herein, the term “liposome” includes any compartment surrounded by a lipid bilayer. The term liposome includes unilamellar vesicles consisting of a single lipid bilayer and generally having a diameter in the range of about 20 to about 400 nm. Liposomes can also be multilayers having a diameter in the range of about 1 μm to about 10 μm. Multilamellar liposomes can consist of several (anywhere from 2 to several hundred) unilamellar vesicles that form one inside the other in decreasing size, creating a multilamellar structure of concentric phospholipid spheres separated by layers of water. Alternatively, multilamellar liposomes can consist of many smaller non-concentric spheres of lipid inside a large liposome. In embodiments, liposomes include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV). In some embodiments, the composition includes liposomes containing any suitable ionizable lipid and neutral lipid, together with the peptides provided herein.

[0116] In some embodiments, the composition comprises lipid nanoparticles (LNPs). The LNP composition is typically on the order of a few micrometers or smaller in size and may include a lipid bilayer. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 1 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 400 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 20 nm to about 100 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 400 μm.In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation having a size of from about 100 nm to about 150 μm.

[0117] The properties of lipid nanoparticles (e.g., LNP without payload or LNP with payload) can depend on their components. For example, lipid nanoparticles containing cholesterol as a structural lipid can have different properties from lipid nanoparticles containing different structural lipids. Similarly, the properties of lipid nanoparticles (e.g., LNP without payload or LNP with payload) can depend on the absolute or relative amounts of their components. For example, lipid nanoparticles containing a higher mole fraction of phospholipid can have different properties from lipid nanoparticles containing a lower mole fraction of phospholipid. The properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0118] Lipid nanoparticles can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be utilized to determine the particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple properties of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0119] The lipid complex compositions provided herein can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the nanoparticle composition, e.g., the particle size distribution of lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. Lipid nanoparticles can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of lipid nanoparticles (e.g., LNP without payload or LNP with payload) can be about 0.10 to about 0.20.

[0120] The zeta potential of lipid nanoparticles (e.g., LNP without payload or LNP with payload) can be used to indicate the electrokinetic potential at the interface of the composition. For example, the zeta potential can account for the surface charge of the nanoparticle composition. Lipid nanoparticles with relatively low positive or negative charges are generally desirable because more highly charged species can interact unfavorably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of lipid nanoparticles can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV, about +15 mV, or about +5 mV to about +10 mV.

[0121] In some embodiments, the payload of the lipid complex composition is an RNA molecule. For example, the payload RNA molecule includes mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, or a combination thereof. In some embodiments, the payload RNA molecule includes more than one mRNA molecule (e.g., at least two mRNA molecules, at least three mRNA molecules, at least four mRNA molecules, or at least five mRNA molecules). In some embodiments, the payload includes at least one sgRNA. In other embodiments, the payload of the lipid complex composition molecule includes an sgRNA molecule and an mRNA molecule.

[0122] In some embodiments, the lipid composition payload includes a gene editing reagent (or gene editing composition), and the gene editing reagent includes a gene editing protein, an RNA molecule, and / or a ribonucleoprotein (RNP). In various examples, the gene editing protein includes a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas protein, MegaTal, Cre recombinase, Hin recombinase, or Flp recombinase. In some embodiments, the RNA molecule includes sgRNA, crRNA, and / or tracrRNA. Thus, in some embodiments, the lipid complex composition payload includes an RNP and an sgRNA. In some embodiments, the RNP can include a Cas protein and an sgRNA, crRNA, or tracrRNA.

[0123] In other embodiments, the RNA can encode a gene editing protein (e.g., an RNA encoding a ZFN, TALEN, Cas protein, Cre recombinase, etc.). Thus, in some embodiments, the lipid complex composition payload comprises an RNA encoding a gene editing protein and an sgRNA. In some embodiments, the lipid complex composition payload can comprise an RNA encoding a Cas protein, and an sgRNA, crRNA, or tracrRNA.

[0124] In some embodiments, the nucleic acid payload of the lipid complex composition is a single-stranded molecule. In some embodiments, the payload can comprise donor DNA. In still other embodiments, the DNA payload can be plasmid DNA or linear DNA. In some examples, the payload can be an RNP and can comprise a Cas protein, and an sgRNA, crRNA, or tracrRNA.

[0125] In certain embodiments, the gene editing payload induces single-stranded or double-stranded breaks in the DNA within the cell. In some embodiments, the gene editing reagent (or gene editing composition) further comprises a repair template polynucleotide. In various embodiments, the repair template comprises (a) a first flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on the other side of the single-stranded or double-stranded break, or (b) a first flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single-stranded or double-stranded break, and a second flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on the other side of the single-stranded or double-stranded break. A non-limiting description of gene editing (including repair templates) using the CRISPR-Cas system is discussed in Ran et al. (2013) Nat Protoc. 2013 Nov;8(11):2281-2308, the entire content of which is incorporated herein by reference. Embodiments with repair templates are not limited to those involving the CRISPR-Cas system.

[0126] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found under accession number Q99ZW2 in the SwissProt database and accession number Q99ZW2.1 in the NCBI database. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of the Cas9 protein amino acid sequence. Another non-limiting example is the Streptococcus thermophilus Cas9 protein, whose amino acid sequence can be found under accession number Q03JI6.1 in the UniProt database. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In certain embodiments, the CRISPR enzyme is Cas9 and can be Cas9 derived from S. pyogenes or S. pneumoniae. In various embodiments, the CRISPR enzyme directs cleavage of one or both strands at the position of the target sequence, such as within and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a CRISPR enzyme that has been mutated relative to the corresponding wild-type enzyme such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence.For example, substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves a single strand). Other examples of mutations that convert Cas9 to a nickase include, but are not limited to, H840A, N854A, and N863A. In aspects of the invention, the nickase can be used for genome editing via homologous recombination.

[0127] In certain embodiments, the payload can include, respectively, a guide sequence that targets the sense and antisense strands of a DNA target, e.g., a Cas9 nickase used in combination with two guide sequences. This combination enables both strands to be nicked and used to induce NHEJ.

[0128] In some further examples, the payload of the lipid composition includes an RNA molecule, and the RNA molecule includes an sgRNA, a crRNA, a tracrRNA, or a combination thereof.

[0129] In some embodiments, the payload of the lipid composition is an immunogen. In some embodiments, the nucleic acid payload of the lipid composition encodes an immunogen. In some examples, the payload nucleic acid of the lipid composition encodes a hemagglutinin (HA) protein or a fragment thereof. In some examples, the nucleic acid payload of the lipid composition encodes ovalbumin or a fragment thereof. In an example, the lipid composition delivers a payload that induces an immune response against a protein encoded by the protein or nucleic acid of the lipid composition in a subject.

[0130] Exemplary transfection-enhancing peptides for use in lipid compositions are provided herein. In some embodiments, such peptides comprise the sequence LLELLESL (SEQ ID NO: 1) and optionally comprise a polycationic nucleic acid-binding moiety. In some embodiments, the peptides comprise ALLELLESL (SEQ ID NO: 2), ELLELLESL (SEQ ID NO: 3), LLELLESLW (SEQ ID NO: 4), and / or LLELLESLY (SEQ ID NO: 5). In some embodiments, the peptides comprise one or more PSYYRYD (SEQ ID NO: 25) or PSYYRGD (SEQ ID NO: 26) sequences and optionally comprise a polycationic nucleic acid-binding moiety.

[0131] As described herein, suitable polycationic nucleic acid-binding moieties include, but are not limited to, polyamines and polybasic peptides, such as those having a length of about 8 to about 20 residues, including, for example, polyarginine, polylysine, polyhistidine, and / or polyornithine sequences.

[0132] The peptides for use in the compositions provided herein are at least 10 amino acids in length. In some embodiments, the peptides are at least 10 to about 100, at least 10 to about 75, at least 10 to about 50, at least 10 to about 40, at least 10 to about 30, or at least 10 to about 20 amino acids in length. In certain embodiments, the peptides are about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 50, about 15 to about 60, or about 15 to about 70 amino acids in length. In some embodiments, the peptides are about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 70, or about 20 to about 80 amino acids in length.

[0133] In some examples, the peptide has at least 80% sequence identity with SEQ ID NO: 6 (GLFEALLELLESLWELLLEA). In other examples, the peptide comprises SEQ ID NO: 6 or a fragment thereof. In other examples, the peptide has at least 85% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 90% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 91% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 92% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 93% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 94% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 95% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 96% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 97% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 98% sequence identity with SEQ ID NO: 6. In other examples, the peptide has at least 99% sequence identity with SEQ ID NO: 6. In other examples, the peptide has 100% sequence identity with SEQ ID NO: 6.

[0134] "Polypeptide fragment" refers to a polypeptide having amino-terminal and / or carboxy-terminal deletions where the remaining amino acid sequence is usually identical to the corresponding positions in the naturally occurring sequence. Fragments are typically at least 5, 6, 8, or 10 amino acids in length, at least 14 amino acids in length, at least 20 amino acids in length, at least 50 amino acids in length, or at least 70 amino acids in length.

[0135] The "percentage of sequence identity" is determined by comparing two sequences optimally aligned in a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (excluding additions or deletions) for the optimal alignment of the two sequences. In embodiments, the percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0136] The terms "identical" or "identity" rate in the context of two or more nucleic acid or polypeptide sequences, when measured using a sequence comparison algorithm or by manual alignment and visual inspection, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or specified region (e.g., over a specified region of the entire polypeptide sequence or an individual domain thereof, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity). In embodiments, the two sequences are 100% identical. In embodiments, the two sequences are 100% identical over the full length of one of the sequences (e.g., the shorter of the two sequences if the sequences have different lengths). In embodiments, identity may refer to the complement of the test sequence. In embodiments, identity is present over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. In embodiments, identity is present over a region that is at least about 50 amino acids or nucleotides in length, or more preferably, over a region that is 100 to 500, 100 to 200, 150 to 200, 175 to 200, 175 to 225, 175 to 250, 200 to 225, 200 to 250, or more amino acids or nucleotides in length.

[0137] Exemplary peptides useful in the formulations provided herein include, but are not limited to, the peptides in Table 1.

[0138] (Table 1) Peptide sequences TIFF2025524562000002.tif99149

[0139] In some embodiments, any of the peptides of SEQ ID NOs: 1-22 may further include an arginine residue or additional arginine residues (e.g., R2, R4, R6, R8, R12) at the N- or C-terminus. In other embodiments, any of SEQ ID NOs: 1-22 may further include a lysine residue or additional lysine (e.g., K2, K4, K6, K8, K12) at the N- or C-terminus. In other embodiments, any of the peptides of SEQ ID NOs: 1-22 may include a plurality of histidine residues (e.g., H2, H4, H6, H8, H12) at the N- or C-terminus. In other embodiments, any of the peptides of SEQ ID NOs: 1-22 may include a plurality of ornithine residues at the N- or C-terminus. In certain embodiments, the sequences of SEQ ID NOs: 1-22 are connected to a plurality of arginine, lysine, histidine, or ornithine residues by a spacer or linker molecule.

[0140] In some embodiments, in addition to a peptide comprising one or more of SEQ ID NOs: 1-5, or a peptide having at least 80% sequence identity to any one of SEQ ID NOs: 6-24, the formulation may include additional transfection enhancers such as cell surface ligand peptides and / or nuclear localization agents such as nuclear receptor ligand peptides. Examples of such transfection enhancers include, but are not limited to, retrovirus-related fusion peptides, insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeting antibodies, lactoferrin, fibronectin, adenovirus penton base, Knob, hexon protein, vesicular stomatitis virus glycoprotein, Semliki Forest virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histone protein, arginine-rich cell-permeable protein, high mobility group protein, and invasin protein, as well as internalin protein, endotoxin, diphtheria toxin, Shigella toxin, melittin, magainin, gramicidin, cyclophilin, defensin, protegrin, tachyplesin, thionin, indolicidin, bacteriocin, drosomycin, apidaecin, cathelicidin, proteins that increase bacterial permeability, nisin, buforin, and fragments thereof, and those disclosed in US Patent Application Publication No. 2018 / 0340188, which is incorporated herein by reference in its entirety.

[0141] Exemplary transfection-enhancing peptides useful in the formulations provided herein include, but are not limited to, peptides comprising one or more sequences of PSYYRYD (SEQ ID NO: 25) and / or PSYYRGD (SEQ ID NO: 26), including the exemplary peptides of Table 2.

[0142] (Table 2) TIFF2025524562000003.tif55146

[0143] In some embodiments, the transfection-enhancing peptide comprises at least two PSYYRYD (SEQ ID NO: 25) and / or PSYYRGD (SEQ ID NO: 26) sequences, or at least three PSYYRYD (SEQ ID NO: 25) and / or PSYYRGD (SEQ ID NO: 26) sequences. In some examples, the transfection-enhancing peptide has at least 80% sequence identity with any one of SEQ ID NOs: 27-36. In other examples, the transfection-enhancing peptide comprises any one of SEQ ID NOs: 27-36, or a fragment thereof. In other examples, the transfection-enhancing peptide has at least 85% sequence identity with any one of SEQ ID NOs: 27-36. In other examples, the transfection-enhancing peptide has at least 90% sequence identity with any one of SEQ ID NOs: 27-36. In other examples, the transfection-enhancing peptide has at least 95% sequence identity with any one of SEQ ID NOs: 27-36. In other examples, the transfection-enhancing peptide has 100% sequence identity with any one of SEQ ID NOs: 27-36. In some embodiments, any one of the peptides of SEQ ID NOs: 27-36 may further comprise additional arginine residues (e.g., R2, R4, R6, R8, R10, R12, R14, R16, R18, R20) at the N or C terminus. In other embodiments, any one of SEQ ID NOs: 28, 30, 32, 34, and 36 may further comprise lysine residues (e.g., K2, K4, K6, K8, K10, K12, K14, K16, K18, K20) at the N or C terminus. In other embodiments, any one of the peptides of SEQ ID NOs: 27-36 may comprise a plurality of histidine residues (e.g., H2, H4, H6, H8, H12) at the N or C terminus. In other embodiments, any one of the peptides of SEQ ID NOs: 27-36 may comprise a plurality of ornithine residues at the N or C terminus. In certain embodiments, the sequences of SEQ ID NOs: 27-36 are connected to a plurality of arginine, lysine, histidine, or ornithine residues by a spacer or linker molecule.

[0144] In some embodiments, the provided lipid composition includes a peptide having and containing LLELLESL (SEQ ID NO: 1), and a peptide containing at least one or more sequences of PSYYRYD (SEQ ID NO: 25) and / or PSYYRGD (SEQ ID NO: 26). In some embodiments, the provided lipid composition includes a peptide having at least 80% sequence identity with SEQ ID NO: 6, and a peptide having at least 80% sequence identity with SEQ ID NO: 27. In other embodiments, the provided lipid composition includes a peptide having at least 80% sequence identity with SEQ ID NO: 6, and a peptide having at least 80% sequence identity with SEQ ID NO: 33. In some embodiments, the lipid composition includes a peptide selected from the peptides of SEQ ID NOs: 6-24, and a peptide selected from the peptides of SEQ ID NOs: 27-36.

[0145] In some embodiments, the endosome-releasing peptide and / or cell surface ligand peptide for use in the provided composition can be linked to a glycosylphosphatidylinositol (GPI) anchor peptide, such as FTLTGLLGTLVTMGLLT (SEQ ID NO: 37), for example.

[0146] In some embodiments, the peptides described herein are directly bound to a nucleic acid binding molecule by a covalent bond or are connected to the binding molecule via a spacer. The terms "spacer" or "linker", used interchangeably herein, refer to a chemical structure that links two molecules to each other as used herein. In some embodiments, the spacer binds to each molecule on different portions of the spacer molecule. In other embodiments, the spacer is a hydrophilic moiety and contains from about 6 to 30 carbon atoms. In other embodiments, the spacer is a polyether, for example, -CH2-O-(CH2-CH2-O-) i CH2-. In other embodiments, the spacer is a hydrophilic polymer, for example, [(gly) i (ser) j k ​It includes these. In these formulas, i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In some embodiments, the spacer is a peptide of the sequence APYKAWK (SEQ ID NO: 38). In other embodiments, the spacer is a sequence that is degraded in vivo by a peptidase.

[0147] The peptides for use in the compositions provided herein are at least 10 amino acids in length. In some embodiments, the peptides are at least 10 to about 100, at least 10 to about 75, at least 10 to about 50, at least 10 to about 40, at least 10 to about 30, or at least 10 to about 20 amino acids in length. In certain embodiments, the peptides are about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 50, about 15 to about 60, or about 15 to about 70 amino acids in length. In some embodiments, the peptides are about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 70, or about 20 to about 80 amino acids in length.

[0148] In some embodiments, the provided lipid composition contains a peptide, and the peptide has a concentration of about 0.001 to about 0.5 mg / mL. In embodiments, the peptide has a concentration of about 0.05 mg / mL to about 0.5 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.001 to about 0.05 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.001 to about 0.1 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.01 to about 0.5 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.01 to about 0.4 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.01 to about 0.3 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.01 to about 0.2 mg / mL. In an example, the lipid composition contains a peptide, and the peptide has a concentration of about 0.01 to about 0.1 mg / mL. In embodiments, the peptide has a concentration of about 0.05 mg / mL to about 0.1 mg / mL. In embodiments, the peptide has a concentration of about 0.05 mg / mL to about 0.2 mg / mL. In embodiments, the peptide has a concentration of about 0.05 mg / mL to about 0.3 mg / mL. In embodiments, the peptide has a concentration of about 0.05 mg / mL to about 0.4 mg / mL. In embodiments, the peptide has a concentration of about 0.1 mg / mL to about 0.5 mg / mL. In embodiments, the peptide has a concentration of about 0.1 mg / mL to about 0.4 mg / mL. In embodiments, the peptide has a concentration of about 0.1 mg / mL to about 0.3 mg / mL. In embodiments, the peptide has a concentration of about 0.1 mg / mL to about 0.2 mg / mL.

[0149] As used herein, the term "ionizable lipid" refers to a lipid having one or more functional groups that can be reversibly ionized (protonated or deprotonated) depending on the pH of the lipid-containing medium. The functional groups can be basic, such as an amino functional group, or acidic, such as a carboxylic acid moiety. One of ordinary skill in the art will recognize that other ionizable functional groups can also be used. An ionizable lipid can contain both a basic moiety and an acidic moiety. Advantageously, the ionizable lipid has an overall positive charge at physiological pH.

[0150] The ionizable lipids described herein refer to lipids having at least one protonatable or deprotonatable group such that the lipid has a positive charge at physiological pH (e.g., pH 7.4) or below pH, and is neutral at a second pH, preferably physiological pH or above. Preferably, the ionizable lipids provided herein have a pKa of protonatable groups in the range of about 5-7, such as about 4-about 11, such as about 4-about 7, such as about 5.5-6.9, when incorporated into a lipid composition, such as a liposome, lipid nanoparticle, or other lipid complex. Ionizable lipids include, for example, amine-containing lipids that can be readily protonated.

[0151] Ionizable lipids include, for example, DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleyloxypropyl)-2-(3-amino-2-hydroxypropyl-oxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyl-oxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-(3-amino-2-hydroxypropyl-oxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-di-lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O’-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-lysine-bis-(O,O’-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O’-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O’-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-Bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-myristoylamino]-butane-2,3-diol, 1,4-bis[(3-oleylamino)propyl]piperazine, L-arginine-bis-(O,O’-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristoylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O’-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxyl-amidoethyltrimethylammonium iodide, 1,4-bis[(3-myristoylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3β-carboxamidoethyleneamine, cholesteryl-3β-oxysuccinamidoethyltrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N’,N’-dimethylaminoethane)carbamoyl]cholesterol, and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitoylamino)propyl]piperazine, L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycineamide, N, 2 ,N 5-Bis(3-aminopropyl)-L-ornithylglycyl-N-(1-heptadecyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornithyl]-N,N-dioleyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl-L-ornithyl]-N-N-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N,N-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornithyl-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl-N-N-dipalmitoyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl-N-N-dipalmitoyl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N,N-dipalmitoyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornithyl-N,N-dimyristoyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl-N-N-dimyristoyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl-N-N-dimyristoyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N,N-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornithyl]-N,N-dilauryl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl]-N-N-dilauryl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithyl]-N-N-dilauryl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dilauroyl-L-glutaminyl]-L-glutamic acid, 3-[N’,N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dioctadec-9-enylpropionamide, 3-[N’,N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dipalmitoylpropionamide, 3-[N’,N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dimyristoylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristoylaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium iodide, [(3-aminopropyl)-bis-(2-oleoyloxyethyl)]methylammonium iodide, [(3-aminopropyl)-bis-(2-palmitoyloxyethyl)]methylammonium iodide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-Dimethylaminopropane, (3-amino-propyl)->4-(3-aminopropylamino)-4-tetradecylcarbamoyl-butylcarbamic acid cholesteryl ester, (3-amino-propyl)->4-(3-aminopropylamino-4-carbamoylbutylcarbamic acid cholesteryl ester, (3-amino-propyl)->4-(3-aminopropylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamic acid cholesteryl ester, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amidotetratri, Fluoroacetate, Spermine-5-carboxyglycine (N'-stearyl-N'-elaidyl) amide tetra trifluoroacetate, Agmatinyl carboxycholesterol acetate, Spermine-5-carboxy-β-alanine cholesterol ester tetra trifluoroacetate, 2,6-Diaminohexanoyl β-alanine cholesterol ester bis trifluoroacetate, 2,4-Diaminobutyroyl β-alanine cholesterol ester bis trifluoroacetate, N,N-Bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tris trifluoroacetate, [N,N-Bis(2-hydroxyethyl)-2-aminoethyl] aminocarboxycholesteryl ester, Stearyl carnitine ester, Palmitoyl carnitine ester, Myristyl carnitine ester, Stearyl stearoyl carnitine ester chloride salt, L-Stearyl stearoyl carnitine ester, Stearyl oleoyl carnitine ester chloride, Palmitoyl palmitoyl carnitine ester chloride, Myristyl myristoyl carnitine ester chloride, L-Myristyl myristoyl carnitine ester chloride, 1,4-Bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)propyl] piperazine, N-(3-Aminopropyl-N,N'-bis-(dodecyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl-N,N'-bis-(oleoyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N,N'-bis-(palmitoyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N,N'-bis-(myristyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N'-methyl-N,N'-(bis-2-oleoyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxyethyl)-piperazinium iodide, N-(3-Aminopropyl)-N'-methyl-N,N'-(bis-2-myristyloxyethyl)-piperazinium iodide, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine, and 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxy-propyl]piperazine, KL22, KL25, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)--octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)) may be selected from the group consisting of.,

[0152] Some embodiments contemplate ionizable lipids described in U.S. Patent Nos. 7,173,154, 9,856,496, and U.S. Patent Application Publication No. 2019 / 0060482 for use in the compositions and methods (the references are incorporated by reference in their entirety).

[0153] In some preferred but non-limiting embodiments, the lipid composition can comprise at least a first ionizable lipid and at least a first neutral lipid, the lipid composition is suitable for forming a complex with nucleic acid under aqueous conditions, and the ionizable lipid has the structure of formula (I): TIFF2025524562000004.tif29128 and its salts, wherein R1 and R2 are independently an alkyl, alkenyl, or alkynyl group having 8 to 30 carbon atoms, an alkyl, alkenyl, or alkynyl group having 8 to 30 carbon atoms, optionally substituted by one or more of an alcohol, amino alcohol, amine, amide, ether, polyether, ester, mercaptan, alkylthio, or carbamoyl group, or R1 is -(CH2) q -N(R6) t R7R8, R3 and R4 are independently hydrogen or an alkyl, alkenyl, or alkynyl group having 8 to 30 carbon atoms, optionally substituted by one or more of an alcohol, amino alcohol, amine, amide, ether, polyether, ester, mercaptan, alkylthio, or carbamoyl group, R5 to R8 are independently hydrogen or an alkyl, alkenyl, or alkynyl group, R9 is hydrogen or an alkyl, alkenyl, or alkynyl group, carbohydrate, or peptide, r, s, and t are 1 or 0 to indicate the presence or absence of the indicated R group. When any of r, s, or t is 1, the nitrogen to which the indicated R group is attached has a positive charge, and at least one of r, s, or t is 1, q is an integer in the range of 1 to 6 (including both end numbers), X v- is an anion, v is the valence of the anion, and A is the number of anions, L is a divalent organic radical capable of covalently sharing two nitrogens, which is (CH2) nwherein n is an integer in the range of 1 to 10 (including both end numbers), and one or more ZR 10 groups are optionally substituted, wherein Z is O or S, and R 10 is hydrogen, or an alkyl, alkenyl, or alkynyl group, (CH2) n or {-(CH2) k -Y-(CH2) m} p -, wherein k and m are independently integers in the range of 1 to 10 (including both end numbers), p is an integer in the range of 1 to 6 (including both end numbers), and Y is O, S, CO, COO, CONR 11 , NR 11 CO, or NR 11 COR 11 N, wherein R 11 is independent of any other R 11 and is hydrogen or an alkyl group, {-(CH2) k -Y-(CH2) m} p is selected from -, One or more CH2 groups of the alkyl, alkenyl, or alkynyl groups of R1 to R 10 can be replaced by O, S, S-S, CO, COO, NR 12 CO, NR 12 COO, or NR 12 CONR 12 , and R 12 is independent of any other R 12 and is hydrogen, or an alkyl, alkenyl, or alkynyl group, The alkyl, alkenyl, or alkynyl groups of R1 to R 12 are optionally substituted by one or more OR 13 , CN, halogen, N(R 13 )2, peptide, or carbohydrate group, and R 13 is independent of other R 13 and is hydrogen, or an alkyl, alkenyl, or alkynyl group, When at least one of R3 and R4 exists as an alkyl group, OR 13 and N(R13 ) It is replaced at both 2 positions.

[0154] Methods for the synthesis of these compounds and the preparation of lipid compositions incorporating them can be achieved by any means known to those skilled in the art, without limitation. Exemplary but non-limiting methods for synthesizing such compounds and methods for forming lipid aggregates incorporating them can be found, for example, in U.S. Patent No. 7,166,745 and PCT Publication No. 00 / 27795, which are hereby incorporated by reference in their entirety as if fully set forth herein.

[0155] In some embodiments of the present lipid aggregates, particularly preferred but non-limiting ionizable lipids used for complex formation have the structural formula IA: TIFF2025524562000005.tif24130 and its salts, wherein R 1 and R 2 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R 3 and R 4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, m is an integer from 1 to 6, and X a - is an anion.

[0156] In some embodiments of the present lipid aggregates, particularly preferred but non-limiting ionizable lipids used for complex formation have the structural formula IB: TIFF2025524562000006.tif24128 and its salts, wherein R 5 and R 8is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R 6 and R 7 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, n is an integer from 1 to 6, X b - is an anion.

[0157] In an embodiment, R 1 and R 2 are independently substituted or unsubstituted alkyl. In an embodiment, R 1 and R 2 are independently unsubstituted alkyl. R 1 and R 2 are independently unsubstituted C1-C 20 alkyl. In an embodiment, R 1 and R 2 are independently unsubstituted C5-C 20 alkyl. In an embodiment, R 1 and R 2 are independently unsubstituted C 10 -C 20 alkyl. In an embodiment, R 1 and R 2 are independently unsubstituted C 12 -C 18 alkyl. In an embodiment, R 1 and R 2 are independently unsubstituted C 14 -C 16 alkyl. In an embodiment, R 1 is unsubstituted C 14 alkyl. In an embodiment, R 2 is unsubstituted C 14 alkyl. In an embodiment, R 1 is unsubstituted C 15 alkyl. In an embodiment, R2 is the unsubstituted C 15 In embodiments, R 1 is the unsubstituted C 16 In embodiments, R 2 is the unsubstituted C 16 In embodiments, R 1 is -(CH2) 13 In an embodiment, R 2 is -(CH2) 13 It is CH3.

[0158] In embodiments, R 3 and R 4 are independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R 3 and R 4 are independently hydrogen.

[0159] In embodiments, R 5 , R 6 , and R 7 are independently hydrogen, substituted or unsubstituted alkyl. In embodiments, R 5 , R 6 , and R 7 are independently hydrogen or unsubstituted alkyl. In embodiments, R 5 , R 6 , and R 7 are independently hydrogen or unsubstituted C1-C 20 In embodiments, R 5 , R 6 , and R 7 are independently hydrogen or unsubstituted C5-C 20 In embodiments, R 5 , R 6 , and R 7 are independently hydrogen or unsubstituted C 10 ~C 20 In embodiments, R 5 , R 6 , and R 7 are independently hydrogen or unsubstituted C 12 ~C 18 In embodiments, R 5 , R6 and R 7 is, independently, hydrogen or unsubstituted C 14 ~C 16 alkyl. In an embodiment, R 5 , R 6 , and R 7 is, independently, hydrogen or unsubstituted C 14 alkyl. In an embodiment, R 5 , R 6 , and R 7 is, independently, hydrogen or unsubstituted C 15 alkyl. In an embodiment, R 5 , R 6 , and R 7 is, independently, hydrogen or unsubstituted C 16 alkyl. In an embodiment, R 5 is unsubstituted C 14 alkyl. In an embodiment, R 7 is unsubstituted C 14 alkyl. In an embodiment, R 5 is -(CH2) 13 CH3. In an embodiment, R 6 is hydrogen. In an embodiment, R 7 is -(CH2) 13 CH3.

[0160] In an embodiment, R 8 is hydrogen or substituted or unsubstituted alkyl. In an embodiment, R 8 is hydrogen.

[0161] In an embodiment, m is an integer from about 1 to 6. In an embodiment, m is an integer from about 1 to 5. In an embodiment, m is an integer from about 1 to 4. In an embodiment, m is an integer from about 1 to 3. In an embodiment, m is an integer from 1 to 6. In an embodiment, m is an integer from 1 to 5. In an embodiment, m is an integer from 1 to 4. In an embodiment, m is an integer from 1 to 3. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6.

[0162] In an embodiment, n is an integer of about 1 to 6. In an embodiment, n is an integer of about 1 to 5. In an embodiment, n is an integer of about 1 to 4. In an embodiment, n is an integer of about 1 to 3. In an embodiment, n is an integer of 1 to 6. In an embodiment, n is an integer of 1 to 5. In an embodiment, n is an integer of 1 to 4. In an embodiment, n is an integer of 1 to 3. In an embodiment, n is 1. In an embodiment, n is 2. In an embodiment, n is 3. In an embodiment, n is 4. In an embodiment, n is 5. In an embodiment, n is 6.

[0163] In one embodiment, R 1 is -(CH2) 13 CH3, R 2 is -(CH2) 13 CH3, R 3 is hydrogen, R 4 is hydrogen, m is 4, and X a is CH3COO - is.

[0164] In one embodiment, R 5 is -(CH2) 13 CH3, R 6 is hydrogen, R 7 is -(CH2) 13 CH3, R 8 is hydrogen, n is 4, and X b - is CH3COO - is.

[0165] Examples of compounds of the structure of Formula I for use as ionizable lipids in the provided lipid compositions and methods include, but are not limited to, the following: TIFF2025524562000007.tif60128TIFF2025524562000008.tif229120

[0166] In some embodiments, ionizable lipids described in U.S. Patent Nos. 9,259,475 and 10,883,118 are contemplated for use in the present compositions and methods (the references are incorporated by reference in their entireties). In some embodiments, such lipids are based on the core of N,N'-disubstituted 2,3-hydroxy-1,4-butanediamine.

[0167] In some non-limiting embodiments, the lipid composition has an ionizable diaminobutane lipid molecule having the structure of Formula II: TIFF2025524562000009.tif26128 and salts thereof, wherein each R 1 is independently C1-C 23 alkyl, C1-C 23 alkenyl, -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, each R 2 is independently -CH2-(CHR 3 ) 1~6 -CH2-NHR 4 or -CH2-(CHR 3 ) 0~6 -CH2-OH, each R 3 is independently H, OH, or NH2, R 4 is H or CH3, and R 10 is H or C1-C8 alkyl.

[0168] In one embodiment, R 1 can be C 14 -C 20 alkyl or monounsaturated C 14 -C 20 -alkenyl. In this and other embodiments, R 2 can be -CH2-(CHR 3 ) 1~6 -CH2-NHR 4 , and R 3 is H or OH. In some embodiments, three or fewer R 3 groups are OH in each R 3 moiety.

[0169] In any of these embodiments, each R 1 may be the same or different, and each R 2 may be the same or different. In a specific embodiment, one or both of the Rs 10 may be H, or one or both of the Rs 10 may be C1-C3 alkyl. For example, one or both of the Rs 10 may be methyl.

[0170] In the above specific embodiments, one or both of the Rs 1 may be C 12 -C 20 alkenyl, and the alkenyl moiety may be cis-alkenyl.

[0171] In a specific embodiment, one or both of the Rs 2 may be -CH2-CHOH-(CHR 3 ) 0~5 -CH2-NHR 4 For example, one or both of the Rs 2 may be -CH2-CHOH-CH2-NH2. In these compounds, R is advantageously C 14~18 alkyl or C 14~18 monounsaturated alkenyl.

[0172] Each R 1 , R 2 , and / or each R 10 may be the same or different, and thus the molecule may be symmetric or asymmetric. In a particular embodiment, R 2 may be C1-C3 alkyl, and / or R 1 may be monounsaturated C 12 -C 20 alkenyl. One or both of the C 1 -C 12 -C 20 alkenyl moieties in R

[0173] The alkenyl moiety, if present, may be cis-alkenyl. Also provided are molecules having the structural formula IIA: TIFF2025524562000010.tif35128 and salts thereof.

[0174] In this structure, each R 1 is independently C1-C 23 alkyl, C1-C 23 alkenyl, -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl. Each R 2 is independently optionally interrupted by up to 2 O atoms, C1-C6 alkyl, or C1-C6 alkenyl. Each R 3 is independently H, C1-C6 alkyl, C1-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C6 alkyl, C5-C7-cycloalkenyl, C5-C7-cycloalkenyl-C1-C6 alkyl, -(CH2) m NR 6 (CH2) n NHR 7 、-(CH2) 2~6 NHR 7 、-(CH2) 3~6 NHC(=NH)NH2((CH2) m O x ) y (CH2) z O x R 8 、or -(CH2) 0~3 Het. Each R 4 is independently H, C1-C6 alkyl, or C1-C6 alkenyl, each R 5 is independently H, an amine protecting group, -(CH2) m NR 6 (CH2) n NHR 7 、-(CH2) 2-6 NHR 7 、-(CH2) 3-6 NHC(=NH)NH2-(CO)C1-C 23 alkyl, -(CO)C1-C 23It may be an alkenyl or a peptide containing 1 to 20 amino acid residues. Advantageously, the peptide contains amino acid side chains with multiple positive charges. Thus, for example, the peptide may contain one or more lysine, arginine, and / or histidine residues. Other amino acids with positive charges, whether naturally occurring or not, can also be used. Thus, for example, ornithine, homoarginine, and other amino acids containing amines, guanidine, imidazole, and other basic heterocycles can be used. Each m can independently be 2 to 5, each n can independently be 2 to 5, while each x can independently be 0 or 1, each y can be 0 to 2, and each z can be 1 to 6. R 6 and R 7 are independently H, an amine protecting group, -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, R 8 can be H, C1-C6 alkyl, or C1-C6 alkenyl, and Het can be a 5- to 7-membered monocyclic basic heterocycle or an 8- to 11-membered bicyclic basic heterocycle.

[0175] The molecules of Structural Formula IIA can be symmetric or asymmetric with respect to each or all of the substituents R 1 ~R 5 , that is, each R 1 can be the same or different, each R 2 can be the same or different, each R 3 can be the same or different, each R 4 can be the same or different, and / or each R 5 can be the same or different.

[0176] In a specific embodiment of Structural Formula IIA, R 2 can be C1-C3 alkyl, and / or R 1 can be monounsaturated C 12 ~C 20 alkenyl. For example, one or both of the C 1 in R 12 ~C 20The alkenyl moiety can be a cis-alkenyl.

[0177] In other embodiments of Formula II, R 3 can be -(CH2) m NR 6 (CH2) n NHR 7 and / or R 5 can be -(CH2) 2~6 NHR 7 In these or other embodiments, m can be 3, n can be 3, and / or R 5 can be -(CH2)3NHR 7 In these and other embodiments, each R 6 and each R 7 can be H.

[0178] In still other embodiments of Formula II, at least one R 6 or R 7 is -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, and the remainder are H. In a specific embodiment, each R 1 can be C 12 -C 20 alkyl or C 12 -C 20 monounsaturated alkenyl, each R 2 can be C1-C3 alkyl, each R 3 can be -(CH2)3NH(CH2)3NH2, each R 4 can be H, and each R 5 can be -(CH2)3NH2. In another specific embodiment, each R 1 can be C 12 -C 20 alkyl or C 12 -C 20 monounsaturated alkenyl, each R 2 can be C1-C3 alkyl, each R 3 can be -(CH2)3NR 6 (CH2)3NHR 7 and each R 4 can be H, and each R 5is -(CH2)3NHR 7 and at least one R 6 or R 7 is -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, and the remainder may be H.

[0179] Specific examples of the compound of Structural Formula II include compounds having the following structures: TIFF2025524562000011.tif50128 and its salts, wherein each R can be, for example, C 12~20 alkyl or C 12~20 alkenyl, but is not limited thereto, and each R' and each R" are independently H, an amine protecting group, -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, but are not limited thereto.

[0180] In these and other embodiments of Structure (Formula IIB), R is C 14 -C 18 alkyl or C 14 -C 18 alkenyl, and / or each R' and each R" are independently H, C 14 -C 18 alkyl or C 14 -C 18 alkenyl.

[0181] Another specific example of the compound of Structural Formula II is a set of compounds having the following structures: TIFF2025524562000012.tif50128 and its salts, wherein, for example, R can be C 12~20 alkenyl, but is not limited thereto, and / or each R' and each R" are independently H, an amine protecting group, -(CO)C1-C 23 alkyl, or -(CO)C1-C 23 alkenyl, but are not limited thereto.

[0182] In these and other embodiments, R can be oleyl, and each R' and each R" can independently be H or oleoyl. In still other embodiments, at least one of R' or R" can be oleoyl and the rest can be H.

[0183] In still other specific embodiments of Structure (II), each R 3 can independently be -(CH2) 2~6 NHR 7 ,-(CH2) 3~6 NHC(=NH)NH2, or -(CH2) 1~3 Het, each R 4 is H, and each R 5 is independently H or a peptide containing 1 to 20 amino acid residues.

[0184] In these and other embodiments, each R 1 can be C 12 ~C 20 alkyl or C 12 ~C 20 alkenyl, each R 2 can be C1-C3 alkyl, and / or each R 5 can be H. In still other embodiments, each R 3 can be -(CH2) 2~6 NH2, -(CH2) 3~6 NHC(=NH)NH2, or each R 3 can be -(CH2) 1~3 Het. In one specific embodiment, Het can be TIFF2025524562000013.tif14128.

[0185] In some embodiments of the present lipid composition, the non-limiting ionizable lipid used in the composition can be a compound having the structure of Formula II below, wherein R is C 14 , C 16 , or C 18 alkyl, or C 14 , C 16 , or C 18 monounsaturated alkenyl: TIFF2025524562000014.tif146128TIFF2025524562000015.tif172128TIFF2025524562000016.tif236106

[0186] In some embodiments, the lipids described in U.S. Patent No. 8,759,499 are contemplated for use in the present compositions and methods (the contents of which are incorporated by reference in their entirety).

[0187] In some non-limiting embodiments, the lipid composition can comprise at least a first ionizable lipid and optionally at least a first neutral lipid, and the lipid composition is suitable for forming a complex with nucleic acids under aqueous conditions, and the ionizable lipid has the structure of formula III: TIFF2025524562000017.tif23148 and its salts.

[0188] In the compound of formula III, X1 and X2 are independently selected from the group consisting of (CH2) n , (CHOH) n , and CONH. X5 and X6 can independently be (CH2) 1~6 . W1 and W2 are independently hydrogen, -OH, -O-(C1-C 30 ) alkyl, -O-(C1-C 30 ) alkenyl, -O-(C1-C 30 ) alkynyl, -NH2-NH(CH2) s CH3-N((CH2) s CH3), -SH, and -NH-NH2. R3 and R6 are independently selected from the group consisting of N, NH, CH, N(CH2) s CH3, (CH) n , (COH) n , CON-, and q = 0-1. R4 and R5 are independently (CH2) n , (CH2-CHOH-CH2) n , (CHOH) nIt may be selected from the group consisting of HNCO, CONH, CO, -O-, -S-, -S-S-, polyamide, and ester bond. L1 and L2 are independently selected from the group consisting of -NH-, -O-, -NHCO-, -CONH-, -OCO-, -COO-, -CO-, -S-, -S-S-, -NHC(O)O-, -OC(O)NH-, -NHCONH-, -NHC(=NH)NH-, -S(O)-, and -SO2-.

[0189] Y is a heterocyclic moiety containing at least one amine or amide moiety. The point of attachment of Y can be a carbon and / or a heteroatom. Examples of suitable heterocyclic moieties include, but are not limited to, piperazine, piperidine, pyridine, pyrrolidine, and imidazole moieties, and derivatives thereof. In a specific embodiment, the heterocyclic moiety is a piperazine ring and the point of attachment is optionally at one or both of the nitrogen atoms. The heterocyclic moiety can be optionally substituted with up to 4 substituents selected from the group consisting of OH, =O, carboxylic acid, ether, polyether, alkylaryl, amino alcohol, amide, straight-chain alkyl, branched alkyl, cycloalkyl, straight-chain alkenyl, branched alkenyl, cycloalkenyl, straight-chain alkynyl, branched alkynyl, primary alkylamine, secondary alkylamine, tertiary alkylamine, quaternary alkylamine, alkenylamine, secondary alkenylamine, tertiary alkenylamine, quaternary alkenylamine, alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternary alkynylamine, amino alcohol, alcohol, ether, polyether, aryl, benzyl, heterocycle, cycloalkyl, alkyl polyamine, alkenyl polyamine, alkynyl polyamine, spermidine, spermine, carboxyspermine, guanidinium, pyridinium, pyrrolidinium, piperidinium, piperazinium, and aminoacyl, and the alkyl, alkenyl, alkynyl, and alkylamine groups are optionally substituted with at least one hydroxyl, or at least one amine, or at least one hydroxyl and at least one amine.

[0190] R1 and R2 can be independently selected from the group consisting of hydrogen, primary alkylamine, secondary alkylamine, tertiary alkylamine, quaternary alkylamine, alkenylamine, secondary alkenylamine, tertiary alkenylamine, quaternary alkenylamine, alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternary alkynylamine, amino alcohol, alkyl polyamine, alkenyl polyamine, alkynyl polyamine, spermidine, spermine, carboxyspermine, guanidinium, pyridinium, pyrrolidinium, piperidinium, piperazinium, aminoacyl, peptidyl, and protein. In the context of the present invention, it will be understood that, unless specifically indicated otherwise, an alkylamine can be an amine containing a short or long alkyl chain. Similarly, an alkenylamine is understood to contain a short or long alkenyl chain, and the same applies to an alkynylamine.

[0191] Z1 and Z2 can be independently selected from the group consisting of linear alkyl, branched alkyl, cycloalkyl, linear alkenyl, branched alkenyl, cycloalkenyl, linear alkynyl, and branched alkynyl, and m, n, p, and s are independently 0 to 6, provided that when m, n, and p are all 0, Y is excluded and R3 is directly bonded to X2.

[0192] Y can have the following cyclic structure: TIFF2025524562000018.tif38128 wherein X3 and X4 can be independently selected from N and CH, and n1 and n2 are independently 1 to 10. Typically, Y is a 6- to 9-membered ring. In an exemplary specific embodiment, both X3 and X4 are N, and both n1 and n2 are 2, that is, Y is an optionally substituted piperazine moiety. This structure can be optionally substituted at 1 to 4 positions as described above for Y.

[0193] In other specific embodiments, Y can have the following cyclic structure: In TIFF2025524562000019.tif38128, n1 and n2 are independently 1 to 10. Typically, n1 + n2 is 3 to 7. Such a cyclic structure may optionally be substituted with 1 to 4 moieties independently selected as described above for Y.

[0194] Examples of lipids are the following structure where both L1 and L2 are NH and X5 and X6 are CH2: TIFF2025524562000020.tif22140 and its salts. In this structure, R1 - R6, W1, W2, X1, X2, Z1, Z2, Y, m, p, and q are as defined above.

[0195] Specific examples of structures of formula IIIA for use as ionizable lipids in the provided compositions and methods include 1,4 - bis[(3 - (3 - aminopropyl) - oleoylamino) - 2 - hydroxypropyl]piperazine, where R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2 - CHOH - CH2, R3, R6 = N, Z1, Z2 = oleoyl, W1, W2 = H, q, p, m = 1, Y = piperazine. Other examples include ((bis(3 - {N - 3 - aminopropyl - N - palmitoyl}amino - 2 - hydroxypropyl) - piperazine)), and other compounds having alkyl groups with lengths varying from C12 - C18, which include, but are not limited to, compounds where R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2 - CHOH - CH2, R3, R6 = N, W1, W2 = H, q, p, m = 1, Y = piperazine, and Z1 and Z2 are both palmitoyl, myristyl, lauryl, or stearyl.

[0196] Other specific examples of compounds of formula III for use herein include, but are not limited to: Compound 3-1: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = palmitoyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-2: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = myristoyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-3: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = lauryl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-4: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = stearyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-5: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = oleoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-6: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = palmitoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-7: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = myristoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-8: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = lauryl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-9: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CHOH-CH2, R3, R6 = N, Z1, Z2 = stearyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-10: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = oleoyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-11: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = palmitoyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-12: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = myristoyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-13: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = lauryl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-14: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = stearyl, W1, W2 = H, q, p, m = 1, Y = piperazine, Compound 3-15: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = oleoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-16: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = palmitoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-17: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = myristoyl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-18: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = lauryl, W1, W2 = OH, q, p, m = 1, Y = piperazine, Compound 3-19: R1, R2 = H, X1, X2 = CH2, R4, R5 = CH2-CH2-CH2, R3, R6 = N, Z1, Z2 = stearyl, W1, W2 = OH, q, p, m = 1, Y = piperazine.

[0197] In the context of the lipids provided, short-chain alkyl groups are typically C1-C6 alkyl, unless otherwise defined. Long-chain alkyl groups are typically C 10 -C 20 alkyl, or C 10 -C 30 alkyl. When not specifically defined, either definition may be used as appropriate. One of ordinary skill in the art will also understand that other derivative groups, including an alkyl moiety, such as an alkoxy moiety, may also contain short-chain and / or long-chain groups in the context as appropriate, unless otherwise defined. An alkenyl group contains at least one cis or trans carbon-carbon double bond and typically has a chain length of C 10 -C 30 . Exemplary alkenyl groups contain one or two cis double bonds where the double bond is disubstituted. An alkynyl group contains at least one carbon-carbon triple bond and typically has a chain length of C 10 -C 30 . Alkyl, alkenyl, or alkynyl groups can be straight-chain or branched. One of ordinary skill in the art will also understand that other derivative groups, including an alkyl moiety, such as an alkoxy moiety, may also contain short-chain and / or long-chain groups in the context as appropriate, unless otherwise defined.

[0198] In some non-limiting embodiments, the ionizable lipids contemplated for use in the compositions and methods are lipid molecules having the structure of Formula IV: TIFF2025524562000021.tif26128 and salts thereof. In this structure, L1 and L3 are independently absent or independently C3-C8 alkyl optionally interrupted by -CO q -, N, O, or -C(O)O-, monounsaturated C4-C8 alkenyl, -CO q C3-C8 alkyl optionally interrupted by N, O, or -C(O)O-, and -CO q - monounsaturated C4-C8 alkenyl, where q is 1 or 2, or R 1 and R 2When it is not H, L1X and L3Y are, independently, -CR 3 =C(R 4 )R 5 and can be. L2 is C4 - C 9 alkylene optionally substituted at up to two positions by OR 12 . X and Y are, independently, L4 - Het, where L4 is C1 - C4 alkylene and Het is a C4 - C 12 heterocycle containing at least one nitrogen atom, L4 - Het, and can be selected from TIFF2025524562000022.tif106128, R 1 and R 2 are, independently, H, C8 - C 20 alkyl, or monounsaturated C8 - C 20 alkenyl and can be. R 3 ~R 8 are, independently, selected from H, -C1 - C6 alkyl, and -C3 - C6 cycloalkyl. In a specific example, R 3 ~R 8 are, independently, H or C1 - C3 alkyl. R 9 is H, -CO - C8 - C 20 alkyl, -CO - monounsaturated C8 - C 20 alkenyl, C8 - C 20 alkyl, or monounsaturated C8 - C 20 alkenyl and can be. As a specific example, when R 9 is C 14 ~C 18 alkyl or monounsaturated alkenyl, R 9 is H, when R 1 and R 2 are not H, when R 9 is not H and R 1 and R 2 are H, and when R 9 is C 14 ~C 18 alkyl or -CO - C 14 ~C18 alkyl, and R 1 and R 2 is H. Advantageously, R 9 The double bond in, if present, is a cis double bond. In these molecules, X and Y can be the same or different, L and L can independently be the same or different, and R 1 and R 2 can be the same or different.

[0199] In certain embodiments, L2 is C4-C 12 In other embodiments, L2 may advantageously be -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p -, wherein m and p are 1 to 6, n is 0 to 6, and m+n+p=2 to 8. In a specific example, L2 is -CH2CH(OR 9 )CH(OR 9 )CH2-.

[0200] In further embodiments, the molecule may contain heterocyclic moieties linked through an amide or carbamate bond, where L1 is -CO- or -CO 2- and X is L4Het, where Het is a heterocycle as defined below. In still further embodiments, R 1 and / or R 2 is not H, the molecule may contain an enamine moiety and L1X is -CR 5 =C(R 3 )R 4 In such enamine moieties, R3 and R5 may together form a C3-C7 carbocyclic ring.

[0201] The molecule of formula IV is a group containing the substituent R 1 , R 2 , L 2~3 and independently may be symmetric or asymmetric with respect to each or all of X and Y, i.e., each R 1may be the same or different, each R 2 may be the same or different, L1 and L3 may be the same or different, R 3 may be the same or different, X and Y may be the same or different. In addition, the structure of L2 need not be symmetric.

[0202] Specific examples of compounds of the structure of Formula IV for use herein include the following: TIFF2025524562000023.tif52128TIFF2025524562000024.tif21093TIFF2025524562000025.tif202128TIFF2025524562000026.tif180128

[0203] In these molecules, each R 1 or R 2 may be, for example, C 14~18 alkyl or C 14~18 alkenyl, but is not limited thereto, and each R 9 may independently be H, -(CO)C 14 ~C 18 alkyl, or -(CO)C 14 ~C 18 alkenyl, but is not limited thereto.

[0204] In some embodiments, the lipids described in U.S. Patent No. 8,034,977 are contemplated for use in the present compositions and methods (the content of which is incorporated by reference in its entirety).

[0205] In some non-limiting embodiments, the lipid composition can comprise at least a first ionizable lipid and optionally at least a first neutral lipid, and the lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, and the ionizable lipid has the structure of formula (V): TIFF2025524562000027.tif33128 or a salt thereof, wherein X is N(R4R5R6) or dialkyl phosphatidyl, or X is TIFF2025524562000028.tif57128.

[0206] In Structural Formula V, Y and Z can independently be selected from the group consisting of alkoxy, alkanoyloxy, alkylamine, alkylurethane, and alkylguanidine. R 1 and R 2 can independently be selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkylamine, alkylamino alcohol, spermine, spermidyl, and carboxyspermine. R 3 can be H or C1-C4 alkyl. R 4 , R 5 , and R 6 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and alkylaryl, provided that at least one of R 4 , R 5 , and R 6 is a long-chain alkyl or alkenyl. The alkyl, alkenyl, aryl, and alkylaryl groups can contain, for example, 6 to 30 carbon atoms, preferably 10 to 18 carbon atoms, but those skilled in the art will recognize that the groups can contain fewer than 6 or more than 30 carbon atoms. W can be a short-chain (C1-C6) alkyl or alkylamino, and R 7 can be a negative charge or a short-chain (C1-C6) alkyl.

[0207] Examples of the compounds of Structural Formula V are a set of compounds having Structural Formula VA: TIFF2025524562000029.tif27128 and its salts, wherein each R is independently C1-C 30 alkyl, alkenyl, or alkanoyl, Z3 and Z4 are independently C1-C6 alkyl, R 1 and R 2is independently selected from the group consisting of hydrogen, C1-C6 alkyl, alkylamine, alkylamino alcohol, spermine, spermidine, and carboxyspermine, R 3 is H or C1-C4 alkyl, R’ is H or TIFF2025524562000030.tif17128.

[0208] Specific examples of the alkylamino alcohol lipids of formula VA include, but are not limited to, the following: TIFF2025524562000031.tif75128 wherein each R is independently C 10 -C 18 alkyl or alkenyl.

[0209] In some embodiments, additional ionizable lipids contemplated for use in the compositions and methods are provided below (Compounds X-1 to X-16), incorporated herein by reference in their entirety, and any others from FIGS. 1 and 2 of Han et al (2021); "An ionizable lipid toolbox for RNA delivery; vol. 12, page 7233. TIFF2025524562000032.tif200142TIFF2025524562000033.tif211146TIFF2025524562000034.tif224145TIFF2025524562000035.tif28143

[0210] The central amine moiety of the lipid according to formula (I), (IA), (IB), (II), (IIA), (IIB), (IIC), (III), (IIIA), (IV), (V), or other N-containing lipid structures described herein can be protonated at physiological pH. Thus, the lipid can have a positive charge or a partial positive charge at physiological pH.

[0211] Those skilled in the art will recognize that although some of the ionizable lipid molecules are shown herein in their neutral (non-protonated) form for convenience, these molecules exist in a partially or fully protonated form in a solution of appropriate pH, and the present invention encompasses all molecules, without limitation, in their protonated, non-protonated, ionized, and non-ionized forms, unless specifically indicated otherwise.

[0212] Advantageously, in addition to the ionizable lipid, the lipid composition comprises one or more neutral co-lipids, and those skilled in the art will recognize that other co-lipids may be used. The neutral lipid can be selected from the group consisting of, for example, sterols or sterol derivatives, phospholipids, or combinations thereof.

[0213] The neutral lipid can be present at about 5 to 60 mol% of the overall lipid formulation. In some embodiments, the neutral lipid is present at about 15 to 50 mol%, such as 25 to 40 mol%. In certain embodiments, the amount of neutral lipid in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% of the overall lipid formulation.

[0214] In some embodiments, the lipid composition comprises neutral lipids, and the neutral lipids comprise cholesterol. In some embodiments, the neutral lipids comprise sterols. In some embodiments, the neutral lipids comprise dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the neutral lipids comprise dipalmitoylphosphatidylethanolamine (DPhPE). In some embodiments, the neutral lipids comprise lysophosphatidylethanolamine (lyso-PE, 1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine). In some embodiments, the neutral lipids comprise lysophosphatidylcholine (lyso-PC, 1-acyl-3-hydroxy-sn-glycero-3-phosphocholine). In some embodiments, the neutral lipids comprise distearoylphosphatidylcholine (DSPC). In some embodiments, the neutral lipids comprise dioleoylphosphatidylcholine (DOPC). In some embodiments, the neutral lipids comprise dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the neutral lipids comprise palmitoyloleoylphosphatidylcholine (POPC). In some embodiments, the neutral lipids comprise palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). In some embodiments, the neutral lipids comprise dipalmitoylphosphatidylethanolamine (DPPE). In some embodiments, the neutral lipids comprise dimyristoylphosphatidylethanolamine (DMPE). In some embodiments, the neutral lipids comprise distearoyl-phosphatidylethanolamine (DSPE). In some embodiments, the neutral lipids comprise 16-O-monomethyl PE. In some embodiments, the neutral lipids comprise 16-O-dimethyl PE. In some embodiments, the neutral lipids comprise 18-1-trans PE. In some embodiments, the neutral lipids comprise 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE). In some embodiments, the neutral lipids comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).In some embodiments, the neutral lipids include any combination thereof.

[0215] Exemplary phospholipids useful in the compositions disclosed herein include dioleoylphosphoethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPhPE), lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (trans DOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), dilinoleoylphosphocholine (DLPC), dimyristoylphosphocholine (DMPC), diundecanoylphosphocholine (DUPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-linolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-linolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE), or any combination thereof, but not limited thereto.,

[0216] Phospholipids useful in the compositions provided herein can be present, for example, in an amount of about 5 mol% to about 20 mol% of the lipid composition formulation. Advantageously, the phospholipids are present in the range of about 1 mol% to about 40 mol%, such as 1 mol% to about 25 mol%. Preferably, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 12 mol%, 14 mol%, 16 mol%, 18 mol%, or 20 mol% of the overall lipid composition formulation, or any amount therebetween.

[0217] In some embodiments, the phospholipids useful in the composition may be present at about 5 mol% to about 15 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 5 mol% to about 10 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 5 mol% to about 12 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 35 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 30 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 25 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 20 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 15 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 10 mol%. In some embodiments, the phospholipids useful in the composition may be present at about 1 mol% to about 5 mol%.

[0218] In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 0.5 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 1 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 2 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 3 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 4 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 5 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 10 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 12 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 15 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 16 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 17 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 18 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 19 mol%. In some embodiments, the amount of phospholipid in the lipid composition formulations disclosed herein is at least about 20 mol%.

[0219] Other neutral lipids that can be advantageously included in the lipid composition formulations provided herein include sterols, or lipids containing a sterol moiety (“sterol derivatives”). As defined herein, a “sterol” is a subgroup of steroids consisting of steroid alcohols. Exemplary sterols and lipids containing a sterol moiety that are useful in the lipid composition formulations provided herein include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, α-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof, but are not limited thereto. In some embodiments, the structural lipid is a sterol. Some of the lipid composition formulations provided herein include a sterol or a sterol derivative. The sterol or sterol derivative can be present at about 5-60 mol% of the overall lipid composition formulation. Advantageously, the sterol or sterol derivative is present at about 15-50 mol%, such as 25-40 mol%. Preferably, the amount of sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% of the overall lipid formulation. Some of the lipid composition formulations provided herein do not include a sterol or a sterol derivative.

[0220] The lipid complex compositions provided herein (e.g., lipid nanoparticles, liposomes, and lipoplexes) can also include one or more compounds and / or compositions, such as cationic polymers such as polyethyleneimine (PEI), polymers of amino acids with a positive charge such as polylysine, polyornithine, and polyarginine, polyamidoamine, poly(β-amino ester), oligoalkylamine, positively charged dendrimers and fragmented dendrimers, cationic β-cyclodextrin-containing polymers (CD-polymers), DEAE-dextran, and the like. The lipid complex compositions can include linear PEI, branched PEI, and / or their derivatives and modified forms such as cyclodextrin-PEI, stearic acid-PEI, aromatic-PEI, histidinyl-PEI, and PEI-PEG. Linear PEI, branched PEI, and their derivatives are commercially available in various molecular weights.

[0221] The lipid complex compositions provided herein (e.g., lipid nanoparticles, liposomes, and lipoplexes) can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, etc.) derived from or purified from exosomes.

[0222] The term "exosome" refers to small membrane vesicles secreted by most cells, including a cell-specific payload of proteins, lipids, and genetic and other biomolecules that are transported to other cells at different locations in the tissue. Exosomes can be considered liposomal particles. Exosomes or lipid mixtures obtained therefrom can be used in combination with other transfection agents or helper lipid mixtures. Exosomes are also referred to as microvesicles, epididymosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, archaeosomes, and oncosomes.

[0223] Examples of lipid components isolated from exosomes include lysophosphatidylcholine (non-limiting examples thereof are C-18, C-16, C-14, and mixtures), lysophosphatidic acid (non-limiting examples thereof are C-18, C-16, and C-14), sphingomyelin, ceramides (non-limiting examples thereof are C-8 to C-24), disaturated phosphatidylcholine (non-limiting examples thereof are DSPC, DPPC, DMPC, and others such as Cn (n = 8 to 25)), diunsaturated phosphatidylcholine-MIX (non-limiting examples thereof are DOPC, DP(db)PC), phosphatidylserine (PS), phosphatidylinositol (PI), disaturated phosphatidylethanolamine (non-limiting examples are DSPE, DPPE, DMPE), diunsaturated phosphatidylethanolamine-MIX (non-limiting examples thereof are DOPE, DP(db)PE), phosphatidylglycerol (PG) (non-limiting examples thereof are C-18 to C-22), cholesterol, and diglycerides such as cardiolipin, but are not limited thereto.

[0224] Also contemplated are any mixtures of combinations of the ionizable lipids, neutral lipids, cationic polymers, exosomes, and / or lipid mixtures isolated from exosomes listed above.

[0225] The lipid compositions provided herein can also include stabilizers such as stabilized lipids. The stabilized lipids can be neutral lipids or can be charged. Stabilized lipids that can be advantageously used in the formulations provided herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyl-oxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. Other stabilized lipids useful in the compositions disclosed herein include, for example, polyglycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers.In an embodiment, the stabilizer may be polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate (“Myrj52”), poly(propylene glycol) 11-block-poly(ethylene glycol) 16-block-poly(propylene glycol) 11, poly(propylene glycol) 12-block-poly(ethylene glycol) 28-block-poly(propylene glycol) 12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (polyoxyethylene (40) stearate), Brij™ S10 (polyoxyethylene (10) stearyl ether), BRIJ™ L4 = polyoxyethylene (4) lauryl ether, BRIJ™ S20 = polyoxyethylene (20) stearyl ether, BRIJ™ S35 = polyoxyethylene (23) lauryl ether, TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate, Tween 20 / polysorbate 80 / tridecyl-D-maltoside in equal ratios, and combinations thereof. In certain compositions, the stabilizer is present at about 0.1-5 mol% of the lipid composition. For example, in some compositions, the stabilizer is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, or any value therebetween of the lipid composition. In other examples, the stabilizer is present at about 0.5 mol% to about 5 mol% of the lipid composition. In other examples, the stabilizer is present at about 0.5 mol% to about 4 mol% of the lipid composition. In other examples, the stabilizer is present at about 0.5 mol% to about 3 mol% of the lipid composition. In other examples, the stabilizer is present at about 0.5 mol% to about 2 mol% of the lipid composition. In other examples, the stabilizer is present at about 0.5 mol% to about 1 mol% of the lipid composition. In other examples, the stabilizer is present at about 1 mol% to about 5 mol% of the lipid composition.In other examples, the stabilizer is present at about 1 mol% to about 4 mol% of the lipid composition. In other examples, the stabilizer is present at about 1 mol% to about 3 mol% of the lipid composition. In other examples, the stabilizer is present at about 1 mol% to about 2 mol% of the lipid composition.

[0226] In some embodiments of the payload-containing lipid composition, the payload is complexed outside the lipid complex (e.g., liposome, lipid nanoparticle). In some embodiments of the nucleic acid-containing lipid composition, the nucleic acid is complexed outside the lipid complex (e.g., liposome, lipid nanoparticle). In some embodiments, the composition has about 20% to about 50% of the nucleic acid complexed outside the lipid complex. In other embodiments, the composition has about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed outside the lipid complex. The external complexation of the nucleic acid can be measured by methods known in the art such as those in Blakney et al. (2019) Gene Therapy 26:363-372.

[0227] In some embodiments of the payload-containing lipid composition, the payload is complexed inside the lipid complex (e.g., liposome, lipid nanoparticle). In some embodiments of the nucleic acid-containing lipid composition, the nucleic acid is complexed inside the lipid complex (e.g., liposome, lipid nanoparticle). In some embodiments, the composition has an encapsulation efficiency of about 75% to about 95%, or about 85% to about 90%. In some examples, the encapsulation efficiency is about 75% to about 100%. In some examples, the encapsulation efficiency is about 75% to about 95%. In some examples, the encapsulation efficiency is about 75% to about 90%. In some examples, the encapsulation efficiency is about 75% to about 85%. In some examples, the encapsulation efficiency is about 75% to about 80%. In some examples, the encapsulation efficiency is about 80% to about 95%. In some examples, the encapsulation efficiency is about 80% to about 90%. In some examples, the encapsulation efficiency is about 80% to about 85%.

[0228] The encapsulation efficiency (EE%) can be measured using a fluorescence plate-based assay with the RiboGreen reagent. This assay measures the amount of mRNA in a sample containing intact LNPs to determine the amount of unencapsulated RNA, as well as in an LNP sample disrupted by Triton X-100 to measure the total RNA. The % encapsulation efficiency was calculated as the difference between the total RNA and the unencapsulated RNA divided by the total RNA.

[0229] The efficiency of encapsulation of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent that is encapsulated after preparation or otherwise associated with the lipid composition relative to the amount provided initially. In some embodiments, the encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the lipid complex composition before and after decomposing the lipid complex composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in solution. In some embodiments of the lipid compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0230] Method for inducing an immune response Provided herein is a method for inducing an immune response in a subject, comprising the step of administering a lipid composition described herein. In some embodiments, the lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 6), at least one peptide, and a nucleic acid molecule comprising a charge (P), and the composition comprises an N / P ratio of 0.01 to 5.0, or 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.0. In some embodiments, the lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6), at least one peptide, and a nucleic acid molecule comprising a charge (P), and the composition comprises an N / P ratio of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, or 0.2. In other embodiments, the lipid composition comprises an N / P ratio of 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0. In some embodiments, the lipid composition comprises an N / P ratio of 0.01 to 1.0, 0.5 to 2.0, or 1.0 to 8.0. In some embodiments, the administered lipid composition further comprises at least one neutral lipid. In some embodiments, the administered lipid composition further comprises a transfection enhancer, such as a peptide comprising SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0231] In some examples, the step of administering the composition comprises systemic or local administration. For example, local administration comprises intramuscular or subcutaneous administration. In some embodiments, the administration comprises administration to the subject's brain, spinal cord, eye (e.g., intravitreal administration), or lymph node (e.g., intranodal administration). In other examples, the administration can comprise ex vivo administration, wherein immune cells (e.g., dendritic cells) are targeted ex vivo. In an example, the subject is a mammalian subject.

[0232] Also provided herein is a method for delivering a lipid composition to target immune cells, the method comprising administering the lipid composition described herein. In some embodiments, the lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 6), at least one peptide, and a nucleic acid molecule comprising a charge (P), and the composition comprises an N / P ratio of 0.01 to 5.0, or 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.0. In some embodiments, the administered lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6), at least one peptide, and a nucleic acid molecule comprising a charge (P), and the composition comprises an N / P ratio of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, or 0.2. In other embodiments, the lipid composition comprises an N / P ratio of 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0. In some embodiments, the lipid composition comprises an N / P ratio of 0.01 to 1.0, 0.5 to 2.0, or 1.0 to 8.0. In some embodiments, the administered lipid composition further comprises at least one neutral lipid. In some embodiments, the administered lipid composition further comprises a transfection enhancer such as a peptide comprising SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0233] Further provided herein is a method for targeting a payload to target immune cells, the method comprising administering the payload-containing lipid composition described herein. For example, immune cells include T cells, B cells, dendritic cells (DCs), T helper cells, cytotoxic T cells (CTLs), natural killer cells (NKs), macrophages including tissue-specific macrophage populations, or combinations thereof. In some examples, immune cells include DCs. In other examples, immune cells include splenic immune cells.

[0234] In some embodiments, the lipid composition targets the subject's immune cells at least 1.2-fold more than targeting the subject's non-immune cells. In some embodiments, the lipid composition targets the subject's immune cells 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold or more than targeting the subject's non-immune cells.

[0235] Relative to a control level, the determined level can be an increased level. As used herein, the term "increased" with respect to a level (e.g., a protein or mRNA level) refers to any % increase above the control level. In various embodiments, the increased level can be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about an 80% increase, at least or about an 85% increase, at least or about a 90% increase, at least or about a 95% increase relative to the control level.

[0236] Relative to the control level, the determined level can be a decreased level. As used herein, the term "decreased" with respect to a level (e.g., protein or mRNA level) refers to any % decrease below the control level. In various embodiments, the decreased level can be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about an 80% decrease, at least or about an 85% decrease, at least or about a 90% decrease, at least or about a 95% decrease relative to the control level.

[0237] For example, in biodistribution studies, female BALB / c mice, 6 - 10 weeks old, received a lipid formulation containing 5 μg of firefly luciferase mRNA via the intramuscular (IM) or intravenous (IV) route, respectively. Four hours after injection, the mice were injected with d - luciferin (150 mg kg-1, intraperitoneally) and imaged using an IVIS Lumina III system (Perkin Elmer) to observe the luciferase signal. To further observe the signal values of specific organs, tissues including the liver, spleen, muscle, and lymph nodes were immediately collected, and the luminescence of the tissue samples was measured by the IVIS Lumina III system. The luminescence intensity in each region of interest (ROI) of the isolated organs was quantified using Living Image 3.0 software (PerkinElmer, Waltham, USA).

[0238] In addition, targeted delivery of immune cells to the spleen includes biodistribution (described above), whereby efficient delivery specifically to the spleen after intravenous administration of luciferase mRNA was confirmed. Targeted delivery was also evaluated using reporter mRNA and flow cytometry analysis of efficiency.

[0239] In some embodiments, a method for delivering a payload to spleen cells in a subject is provided, the method comprising: (i) mixing at least one ionizable lipid, at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), and the payload to form a lipid complex; and (ii) administering the payload-containing lipid complex to the subject. In some embodiments, a method for delivering a nucleic acid to spleen cells in a subject is provided, the method comprising: (i) mixing at least one ionizable lipid, at least one peptide, wherein the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6), and the payload to form a lipid complex; and (ii) administering the lipid complex to the subject. In some embodiments, at least one neutral lipid is mixed with at least one ionizable lipid and at least one peptide to form a lipid complex. In some embodiments, the payload is a nucleic acid, such as an RNA or DNA molecule. In other embodiments, the payload comprises a peptide or polypeptide molecule.

[0240] In a further example, a method for expressing a protein in spleen tissue in a subject is provided, the method comprising administering a lipid composition (lipid complex) to the subject.

[0241] Method of preparation In some embodiments, a method for preparing a population of lipid formulations containing nucleic acid payload molecules is provided, the method comprising: (a) mixing a nucleic acid payload with a peptide, the peptide comprising the sequence LLELLESL (SEQ ID NO: 1), in an aqueous solution; (b) injecting a lipid solution containing an ionizable lipid into the aqueous solution, the injecting step comprising extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (c) generating a population of lipid formulations complexed with the nucleic acid payload. In some embodiments, the peptide is in an ethanol solution when added. In some embodiments, the lipid solution of (b) further comprises at least one neutral lipid. In some embodiments, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 6-24. In some embodiments, step (a) further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0242] In some embodiments, a method for preparing a population of lipid formulations containing a nucleic acid molecule payload is provided, the method comprising: (a) contacting a peptide comprising the sequence LLELLESL (SEQ ID NO: 1) with a lipid phase comprising an ionizable lipid; (b) contacting the components of step (a) with a nucleic acid payload in an aqueous solution; (c) mixing the components of step (b) by extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (d) generating a population of lipid formulations complexed with the nucleic acid payload. In some embodiments, the peptide and the lipid are in an ethanol solution when mixed with an aqueous solution containing the nucleic acid molecule. In some embodiments, the lipid phase of (a) further comprises at least one neutral lipid. In some embodiments, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 6-24. In some embodiments, step (a) further comprises contacting the components with a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0243] In some embodiments, the preparation method generates a population of lipid nanoparticles having payload molecules encapsulated therein. In other embodiments, the preparation method generates a population of liposomes having a payload encapsulated therein. In some embodiments, the preparation method generates a population of lipid nanoparticles having payload molecules complexed outside the lipid nanoparticles. In other embodiments, the preparation method generates a population of liposomes having payload molecules complexed outside the liposomes. In some embodiments, the payload is a nucleic acid, such as an RNA or DNA molecule. In other embodiments, the payload comprises a peptide or polypeptide molecule.

[0244] Kit In one aspect, a kit comprising a lipid composition is provided. In embodiments, the kit comprises at least one ionizable lipid, at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), and a reagent. In some embodiments of the kit, the peptide comprises at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the kit further comprises at least one neutral lipid. In embodiments, the lipid composition in the kit is suitable for delivery to a subject (e.g., by topical injection).

[0245] The present invention also provides packages and kits comprising a pharmaceutical composition for use in the methods of the present invention. The kit can comprise one or more containers selected from the group consisting of bottles, vials, ampoules, blister packs, and syringes. The kit can further comprise instructions for use in treating and / or preventing a disease, condition, or disorder of the present invention, one or more syringes, one or more applicators, or one or more of the sterile solutions suitable for reconstituting the pharmaceutical composition of the present invention.

[0246] In some examples, a kit comprising a lipid composition provides that the ionizable lipid and the neutral lipid are in a separate container from the peptide. In some examples, a kit comprising a lipid composition provides that the ionizable lipid and the neutral lipid are in the same container as the peptide.

Examples

[0247] The following examples illustrate certain specific embodiments of the invention and are not intended to limit the scope of the invention. The embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are merely intended to illustrate the embodiments and should not be construed as limiting the scope herein. The contents of all references cited throughout this application as well as published patents and patent applications are hereby incorporated by reference into this specification.

[0248] Example 1 A lipid-peptide complex formulation having at least one ionizable lipid and at least one peptide was screened and evaluated by various in vivo functional tests using the RNA payload of the complex. Performance and transfection efficiency analysis included responses to payload delivery, biodistribution, cellular uptake, and expression of the protein encoded by the payload. The mRNAs used included those encoding firefly luciferase (fLuc), Cre recombinase, GFP, and influenza virus hemagglutinin (HA).

[0249] Using standard complexation or lipid nanoparticle formation procedures, at least one ionizable lipid, neutral lipid, and peptide SEQ ID NO: 6 were complexed with the mRNA payload. As exemplified herein, the formulations tested varied, for example, in the concentration of the peptide, N / P ratio, and / or the type of ionizable lipid.

[0250] (Table 3) Exemplary lipid complex formulations TIFF2025524562000036.tif81128

[0251] (Table 4) Exemplary lipid complex formulations TIFF2025524562000037.tif66149

[0252] (Table 5) Exemplary lipid complex formulations TIFF2025524562000038.tif73149

[0253] The concentration of the ionizable lipid in the formulations of Table 3 was in the range of 0.8 mg / l to 2.0 mg / l, and all of the formulations in Tables 3 - 5 included one or two neutral lipids. The type of ionizable lipid refers to the structural formula provided herein.

[0254] The formulations in the examples were formulated using reverse evaporation or microfluidization. Lipids were weighed, dissolved in chloroform, and subsequently evaporated on a rotary evaporator. The lipid thin film was hydrated with water. In some situations, the liposome preparation was passed through a microfluidizer. The lipid formulation was incubated with the peptide solution overnight and stored at 4 °C until use.

[0255] For complex formation with nucleic acids, the lipid formulation and nucleic acid were diluted in buffer, mechanically mixed by pipetting and / or vortexing, and incubated at room temperature for 10 - 20 minutes prior to delivery to the murine model or cells.

[0256] Female BALB / c mice, 6 - 10 weeks old, were purchased from The Jackson Laboratory and acclimatized for 7 days prior to the study.

[0257] Example 2 Firefly luciferase mRNA was complexed with each lipid - peptide formulation. Using intravenous tail vein injection in a total volume of 100 μL, mice were injected with 5 μg of the fLuc mRNA - formulated lipid complex. Four hours after injection, the mice were anesthetized with isoflurane and imaged 10 minutes after an intraperitoneal injection of 100 μL of Rediject D - luciferin (Perkin Elmer). Bioluminescence images were quantified in vivo and ex vivo using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0258] As shown in Figures 1A - 1C, intravenous administration of various lipid complex - peptide formulations resulted in luciferase expression in the spleen. Formulations LP4, LP5, and LP6 all contain the same ionizable lipid and differ only in the amount of peptide. The presence of the peptide in the formulation significantly improved the expression of the payload mRNA in spleen tissue compared to the lipid formulation without peptide. For example, in Figure 1B, the flux for LP4 (without peptide) is to be compared with the fluxes for LP5 and LP6.

[0259] Formulations LP7, LP8, LP9, and LP10 contain the same ionizable lipid and differ only in the amount of peptide. The overall expression in the spleen was lower in the formulation shown in Figure 1C compared to the formulations shown in Figures 1A and 1B, but the presence of the peptide significantly improved the expression of luciferase mRNA in spleen tissue for the lipid formulation based on Formula VA. For example, in Figure 1C, the flux for LP7 (without peptide) is to be compared with the flux for the formulation.

[0260] The in vivo delivery performance of formulations LP11 - LP22 having an N / P ratio in the range of 0.1 - 5 was evaluated after intravenous administration. The spleen flux results are shown in Figure 2. The results of the formulations were compared with the results obtained using the mRNA complexed formulation LP11 without the peptide of SEQ ID NO: 6 ("LP11 - without peptide" in Figure 2).

[0261] After intramuscular administration of formulations LP11 - LP22 having an N / P ratio in the range of 0.1 - 5, the in vivo delivery performance was evaluated after intramuscular administration. The luciferase signal was localized in the injection area (muscle), and the luciferase flux quantification results are shown in Figure 3. As described above, formulation LP11 without the peptide of SEQ ID NO: 6 ("LP11 - without peptide" in Figure 3) was used as a control. No significant fLuc expression was observed in other organs up to 72 hours after intramuscular injection of the formulation, and highly specific non - liver targeting of the formulation was confirmed.

[0262] The N / P ratio was varied in the fLuc mRNA - complexed LP11 and LP12 formulations, and the effects on spleen delivery and biodistribution after intravenous injection were evaluated. As shown in Figure 4A, low N / P ratios up to 0.125 using these formulations were effective in achieving fLuc mRNA expression in the spleen. These formulations also resulted in specific delivery to the spleen rather than the liver or lung (Figure 4B).

[0263] Example 3 We used tdTomato reporter mice to analyze spleen cell populations targeted by lipid-peptide formulations. A schematic diagram of the workflow and analysis is shown in Figure 5. Cre mRNA was complexed with LP11 and LP12 formulations, and the complexes were administered intravenously to Ai14 mice. Five days after injection, the animals were sacrificed and the spleens were harvested.

[0264] For splenocyte analysis, single-cell suspensions were prepared by passing spleens through a 70 μm cell strainer. Subsequently, blood cells were lysed with ACK lysis buffer and washed twice with PBS. Splenocytes were divided into two staining groups and stained with fluorescent antibodies against CD8 (APC), CD4 (PE), and B220 (FITC) or F480 (APC), CD11b (FITC), and CD11c (PE). Fluorescence of BDK-CART transfectants within the subpopulations was detected on an LSR-II.UV (BD Biosciences) using the Pacific Blue channel.

[0265] As shown in Figure 6, the Cre mRNA-containing LP11 formulation delivered functional Cre mRNA to the splenic dendritic cell population. Delivery to splenic dendritic cells was significantly greater than delivery to splenic B and T cell populations. A similar pattern was observed when GFP mRNA was used for delivery.

[0266] The performance of LP11 and LP12 formulations in splenocyte delivery was compared to that of LP11 and LP12 formulations without the peptide of SEQ ID NO:6 ("LP11-no peptide" and "LP12-no peptide" in Figures 7A and 7B). The lipid formulations were complexed with a Cre mRNA-formulated lipid composition and administered intravenously to Ai14 mice. Five days after administration, splenocytes were prepared as described above and labeled with fluorescent antibodies against various immune cell types. A flow cytometry gating strategy was used to identify cell markers versus tdTomato expression.

[0267] Both the LP11 and LP12 formulations were more effective in delivering Cre mRNA to all immune cell types in the spleen (as shown by tdTomato expression) than either the LP11 or LP12 formulations lacking the peptide of SEQ ID NO: 6 (Figure 7A). As also shown in Figure 7A, both the LP11 and LP12 formulations were significantly more effective in delivering mRNA to dendritic cells compared to other immune cells in the spleen. Figure 7B shows representative flow cytometry gating results for the dendritic cell population (MHCII / CD11c) versus tdTomato expression.

[0268] Firefly luciferase (fLuc) mRNA was formulated with the LP11 formulation at various N / P ratios. Similarly, fLuc mRNA was formulated with an LP11 formulation lacking the peptide of SEQ ID NO: 6 at the same N / P ratios. The mRNA-containing compositions were administered via intravenous (IV) injection and imaged 4 hours post-injection as described in Example 2. Figures 8A and 8B show exemplary results for the LP11 formulation and the LP11 formulation lacking the peptide having N / P ratios from 0.25 to 4.0. At N / P ratios of 1.0 to 4.0, the LP11 formulation showed enhancement compared to that of the LP11 formulation lacking the peptide.

[0269] In a repeated dosing experiment, two doses of a composition of fLuc mRNA formulated with the LP11 formulation at N / P 2 were administered via IV injection to 4 mice with a 2-week period between doses. The mice were imaged 4 hours post-injection as described in Example 2. The bioluminescence intensity images showed that the administered formulation specifically delivered the fLuc mRNA payload to the spleen. Total flux (photons / second, p / s) from the expressed fLuc was calculated using the bioluminescence images in the region of interest. The average total flux measured after the first injection was 1.103×10 9 p / s, and after the second injection, it was 1.072×10 9 p / s. Control mice receiving a single dose showed a total flux of 1.103×10 9 p / s. Thus, the formulation enables repeated dosing and prime-boost dosing.

[0270] Example 4 The intracellular endosomal uptake and release of the nucleic acid-containing lipid complex were examined using the workflow depicted in FIGS. 9 and 11. For this analysis, HEK293 cells were transfected with the Cy5-labeled mRNA complexed LP formulation or the LP11 formulation without the peptide of SEQ ID NO: 6 and incubated at 37° C. and 5% CO2. After 15, 30, 60, and 90 minutes, the cells were fixed with 4% formaldehyde and counterstained with DAPI to identify the cell nuclei. A confocal laser scanning microscope (Zeiss) was used for the fluorescence images.

[0271] Exemplary results of the uptake of the mRNA-lipid complex are shown in FIG. 10A, where the red fluorescence indicates Cy5-mRNA and the blue fluorescence indicates DAPI. The quantification of cell uptake over time is shown in FIG. 10B. The LP11 formulation internalized at an earlier time relative to the peptide-free LP11 formulation and showed more efficient cell uptake for the complete LP11 formulation.

[0272] To examine the endosomal escape of the complex, HEK293 cells were transfected with the Cy5-labeled mRNA complexed LP formulation or the LP11 formulation without the peptide of SEQ ID NO: 6 and incubated at 37° C. and 5% CO2 for 3 hours. Prior to fixation, 50 nM of LysoTracker™ Green DND-26 (Invitrogen) was added to the cells for 30 minutes to identify the late endosomes in lysosomal degradation (FIG. 11). The cells were then fixed, counterstained with DAPI as described above, and images were acquired using a confocal laser scanning microscope.

[0273] Exemplary results of endosomal escape of the mRNA-lipid complex are shown in Figure 12, where red fluorescence indicates Cy5-mRNA (the two left panels), green fluorescence indicates a late endosome marker (the two middle panels), and blue fluorescence indicates DAPI (the two right panels). The LP11 formulation showed more efficient endosomal escape than the peptide-free LP11 formulation. As seen in the merged images (Figure 12, the two right panels), much less mRNA was released from LysoTracker staining and appeared using the complete LP11 formulation compared to the peptide-free LP11 formulation.

[0274] Example 5 Immunogenicity is defined as the ability of a substance (e.g., a vaccine) to induce an adaptive immune response. Humoral and cell-mediated immunity are two types of adaptive immune responses that enable the body to defend itself in a targeted manner against foreign substances (antigens). The humoral immune response is an antibody-mediated response that occurs when an antigen is detected in the body. This mechanism is mainly driven by B cells that produce antibodies after detection of a specific antigen. Unlike humoral immunity, cell-mediated immunity is antibody-independent. Cell-mediated immunity is mainly driven by the release of mature T cells and various cytokines (e.g., IFN-γ) in response to an antigen.

[0275] The humoral and cell-mediated immunogenicity of an antigen administered via mRNA encoding the antigen formulated with a lipid composition was evaluated to demonstrate the potential of such formulations for vaccine applications. The workflow and test compositions for the immunogenicity study are shown in Figure 13.

[0276] Lipid-mRNA complexes of an H10N8 influenza hemagglutinin (HA) mRNA complexed LP11 or LP12 formulation were prepared. The mRNA-containing complexes were administered to BALB / c mice either intramuscularly (IM) or intravenously (IV) according to a prime-boost immunization schedule at 4-week intervals. Figure 13 shows the immunization schedule, the mRNA doses and routes of complex administration, and the subsequent assays performed to evaluate humoral and cellular immune responses. The mRNA doses shown in Figure 13 are related to those shown in Figures 14-15 as follows: 0.02 μg is 0.01 mg / kg (mRNA amount / mouse body weight), 1 μg is 0.05 mg / kg, 5 μg is 0.25 mg / kg, 10 μg is 0.5 mg / kg, and 20 μg is 1 mg / kg.

[0277] At 3 weeks after the prime injection and 2 weeks after the booster injection (6 weeks in total), serum IgG antibodies binding to the recombinant HA protein were detected in mouse sera by enzyme-linked immunosorbent assay (ELISA). The HA-specific IgG responses after administration of the composition are shown in Figure 14A for IM administration and Figure 14B for IV administration. The results of HA-specific IgG are shown for serum samples at 3 weeks and 6 weeks from each mouse for each composition administered.

[0278] To evaluate the HA antigen-specific cellular response by interferon-γ (IFN-γ), splenocytes were isolated from the spleen at the end of the prime-boost immunization schedule (6 weeks). HA-specific peptide stimulation was used for T cell activation in in vitro culture, and the released IFN-γ was detected by enzyme-linked immunosorbent spot (ELISPOT) assay. Exemplary results are shown in Figures 15A-15C.

[0279] Delivery of HA mRNA using both the LP11 and LP12 formulations resulted in antigen-specific humoral and cellular immune responses for all doses exemplified, even at the lowest dose tested.

[0280] The invention has been described in conjunction with the forms of carrying out the invention, but the foregoing description is not intended to limit, but rather to illustrate, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0281] The patents and scientific literature referred to herein establish knowledge available to those of ordinary skill in the art. All references cited herein, such as U.S. patents, U.S. patent application publications, PCT patent applications designating the United States, published foreign patents and patent applications, are hereby incorporated by reference in their entirety. Submissions to GenBank and NCBI indicated by accession numbers cited herein are hereby incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0282] The invention has been specifically shown and described with reference to its preferred embodiments, but those of ordinary skill in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A composition for delivering a payload to spleen cells, At least one ionizable lipid, At least one peptide, wherein the peptide contains LLELLESL (SEQ ID NO: 1) and is present at a concentration of less than 1.0 mg / mL, A composition comprising at least one payload molecule.

2. The composition according to claim 1, wherein the concentration of the peptide is about 0.05 mg / mL.

3. The composition according to claim 1 or 2, wherein the payload molecule is a nucleic acid.

4. The composition according to claim 3, wherein the at least one ionizable lipid comprises a charge (N), the nucleic acid comprises a charge (P), and the lipid complex comprises an N / P ratio of 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.

0.

5. A composition, At least one ionizable lipid containing an electric charge (N), At least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 1), A payload comprising at least one nucleic acid, wherein the nucleic acid comprises at least one payload comprising a charge (P), The composition comprises an N / P ratio of 0.01 to 0.2, or 0.05 to 0.5, or 0.1 to 1.0, or 0.5 to 2.0, or 1.0 to 5.

0.

6. A composition, At least one ionizable lipid containing an electric charge (N), At least one endosomal-release peptide, A payload comprising at least one nucleic acid, wherein the nucleic acid comprises at least one payload comprising a charge (P), The composition comprises an N / P ratio of about 0.01 to about 0.

5.

7. The composition according to claim 1, 5, or 6, further comprising at least one neutral lipid.

8. The composition according to claim 1, 5, or 6, wherein the peptide has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

9. The composition according to claim 1, 5, or 6, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 24.

10. The composition according to claim 1, 5, or 6, wherein the lipid complex further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO:

26.

11. The composition according to claim 1, 5, or 6, wherein the peptide is concentrated at a concentration of about 0.001 to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL.

12. The composition according to claim 1, 5, or 6, wherein the payload comprises an RNA molecule.

13. The composition according to claim 12, wherein the RNA molecule comprises one or more mRNA molecules.

14. The composition according to claim 12, wherein the RNA molecule comprises mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or a combination thereof.

15. The composition according to claim 14, wherein the RNA molecule comprises an sgRNA molecule.

16. The composition according to claim 12, wherein the RNA molecule comprises an sgRNA molecule and an mRNA molecule.

17. The composition according to claim 1, 5, or 6, wherein the payload further comprises a protein.

18. The composition according to claim 5 or 6, wherein the nucleic acid encodes an immunogen.

19. The composition according to claim 5 or 6, wherein the nucleic acid encodes hemagglutinin (HA) or ovalbumin.

20. The composition according to claim 1, 5, or 6, wherein the ionizable lipid includes a lipid comprising any one of formulas (I), (II), (III), (IV), or (V), or a lipid comprising a combination thereof.

21. The composition according to claim 1, 5, or 6, wherein the ionizable lipid includes a lipid according to formula (IA) or formula (IB), or a combination thereof.

22. The composition according to claim 1, 5, or 6, wherein the ionizable lipid includes a lipid according to formula (IIA), formula (IIB), or formula (IIC), or a combination thereof.

23. The aforementioned neutral lipids include cholesterol, sterols, dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPPC), palmitoyloleoylphosphatidylethanolamine (POPE), and geo The composition according to claim 7, comprising oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), or a combination thereof.

24. The composition according to claim 1, 5, or 6, wherein the composition comprises a group of lipid nanoparticles.

25. The composition according to claim 24, wherein the lipid nanoparticles include a diameter of about 20 nm to about 1 μm.

26. The composition according to claim 1, 5, or 6, wherein the composition comprises a group of liposomes.

27. The composition according to claim 1, 5, or 6, wherein the composition is intended for administration to a subject via intramuscular, subcutaneous, intravitreous, cerebral, or spinal cord administration.

28. A method for targeting a payload to target immune cells, comprising the step of administering the composition according to any one of claims 1, 5, or 6 to the target.

29. The method according to claim 28, wherein the immune cells include T cells, B cells, dendritic cells (DCs), T helper cells, cytotoxic T cells (CTLs), natural killer cells (NKs), macrophages, or a combination thereof.

30. The method according to claim 28, wherein the immune cells include splenic immune cells.

31. The method according to claim 30, wherein the immune cells include dendritic cells.

32. A method for preparing a group of lipid formulations containing payload molecules, (a) A step of mixing the payload molecule with a peptide, which is a peptide containing LLELLESL (SEQ ID NO: 1), in an aqueous solution. (b) A step of injecting a lipid solution containing ionizable lipids into the aqueous solution, the step of injecting including extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing, (c) A method comprising the step of generating a group of lipid formulations complexed with the payload molecule.

33. The method according to claim 32, wherein the lipid solution in (b) further comprises at least one neutral lipid.

34. A method for preparing a group of lipid formulations containing payload molecules, (a) A step of contacting a peptide containing LLELLESL (SEQ ID NO: 1) with a lipid phase which contains ionizable lipids, (b) A step of bringing the components of step (a) into contact with the payload in an aqueous solution, (c) A step of mixing the components of step (b) by extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing, (d) A method comprising the step of generating a group of lipid formulations complexed with the payload molecule.

35. The method according to claim 34, wherein the lipid phase in (a) further comprises at least one neutral lipid.

36. The method according to any one of claims 32 to 35, wherein the peptide has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

37. The method according to any one of claims 32 to 35, wherein the payload is a nucleic acid molecule.

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