Lipid compositions for in vivo delivery

Abstract

Provided herein are compositions, methods, and kits for inducing an immune response in a subject. In aspects, lipid compositions are described comprising at least one ionizable lipid comprising a charge (N), at least one peptide, and a nucleic acid molecule comprising a charge (P). In aspects, methods are provided for delivering a payload to an immune cell using a lipid composition comprising at least one ionizable lipid, at least one endosome releasing peptide, and a payload.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Application No. 63 / 510,733, filed on June 28, 2023, and U.S. Provisional Application No. 63 / 357,584, filed on June 30, 2022, the contents of which are hereby expressly incorporated by reference in their entirety, as if fully set forth herein.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ST.26 XML format and is hereby incorporated by reference in its entirety. The XML file, created on June 28, 2023, is named TP109375WO1_SL.XML and is 34,000 bytes in size. Technical Field

[0005] The present invention is in the field of lipid compositions and formulations useful for delivering one or more biologically active agents to cells (eg, immune cells), and methods and kits for their use. Background Art

[0006] Without using typical biomolecule targeting techniques, it remains a challenge to design lipid compositions and formulations that can target payloads to specific organs, tissues or cell types. It is highly desirable to deliver biologically active payloads to specific tissues and / or cell types, particularly to improved compositions and methods for immune system tissues and cells. Compositions and methods that address this and other needs in the art are provided herein. Summary of the invention

[0007] In various aspects, provided herein are methods for delivering a payload to a spleen cell of a subject, the methods comprising: (i) providing a lipoplex comprising at least one ionizable lipid; at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 1); and at least one payload molecule; and (ii) administering the lipoplex to the subject. In some embodiments, the lipoplex further comprises at least one neutral lipid.

[0008] In some embodiments, the peptide has at least 80% sequence identity to 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.

[0009] In some embodiments, the concentration of the peptide is about 0.001 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.001 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.001 mg / mL. In other embodiments, the concentration of the peptide is about 0.05 mg / mL. In other embodiments, the concentration of the peptide is about 0.5 mg / mL.

[0010] In certain embodiments, the payload is a nucleic acid molecule. In some embodiments, at least one ionizable lipid comprises a charge (N), the payload nucleic acid comprises a charge (P), and the lipoplex has 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.

[0011] 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.

[0012] In some embodiments, the payload comprises an RNA molecule. In some embodiments, the RNA molecule comprises 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.

[0013] In other embodiments, the payload further comprises a protein.

[0014] In some embodiments, the nucleic acid payload encodes an immunogen. In some embodiments, the nucleic acid payload encodes hemagglutinin (HA) or ovalbumin.

[0015] In some embodiments, the ionizable lipids include lipids according to formula (I), formula (II), formula (III), formula (IV), or formula (V), or a combination thereof. In some embodiments, the ionizable lipids include at least one lipid according to formula (I). In some embodiments, the ionizable lipids include at least one lipid according to formula (II). In some embodiments, the ionizable lipids include at least one lipid according to formula (III). In some embodiments, the ionizable lipids include at least one lipid according to formula (IV). In some embodiments, the ionizable lipids include at least one lipid according to formula (V).

[0016] 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).

[0017] 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).

[0018] In some embodiments, the 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 (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 (trans-DOPE) or a combination thereof.

[0019] In other embodiments, the lipid complex comprises a liposome. In some embodiments, the lipid complex comprises a lipid nanoparticle. In some embodiments, the lipid complex comprises a population of lipid nanoparticles, wherein the nanoparticle has a diameter of about 20 nm to about 1 μm. In some embodiments, the nanoparticle has a diameter of about 10 nm to about 900 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 800 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 700 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 600 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 500 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 400 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 300 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 200 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 100 nm. In some embodiments, the nanoparticle has a diameter of about 20 nm to about 50 nm.

[0020] In embodiments, administration includes systemic administration or local administration. In some embodiments, local administration includes intramuscular administration or subcutaneous administration. In embodiments, administration includes intravenous administration. In embodiments, administration includes administration to the brain, spinal cord, eye or lymph node of the subject.

[0021] In some embodiments, the subject comprises a mammalian subject. In some embodiments, the subject comprises a human.

[0022] In some embodiments, the payload is delivered to dendritic cells of the spleen. In some embodiments, the lipoplex targets the spleen cells of the 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 administering a lipoplex comprising an ionizable lipid and the payload without the peptide.

[0023] Provided herein are methods for expressing a protein in spleen tissue of a subject, the methods comprising administering to the subject via systemic administration a lipid complex described herein.

[0024] In various aspects, provided herein are compositions for delivering a payload to a splenocyte. Provided herein is a composition having at least one ionizable lipid; and at least one peptide, wherein the peptide comprises 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 concentration of the peptide is about 1.0 mg / mL. In other embodiments, the concentration of the peptide is about 0.05 mg / mL.

[0025] Also provided herein are compositions comprising at least one ionizable lipid having a charge (N); at least one peptide, wherein the peptide, wherein the peptide comprises 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 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 is the ratio of positively charged groups (including amine (N) groups) to negatively charged groups (including phosphate (P) and peptide groups).

[0026] Also provided herein is a composition comprising: at least one ionizable lipid having a charge (N); at least one endosomally released peptide; and at least one payload comprising a nucleic acid having a charge (P), wherein the composition has an N / P ratio of about 0.01 to about 0.5.

[0027] 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.

[0028] In some embodiments, the concentration of the peptide is about 0.001 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.001 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.05 mg / mL to about 0.5 mg / mL. In some embodiments, the concentration of the peptide is about 0.001 mg / mL. In other embodiments, the concentration of the peptide is about 0.05 mg / mL. In other embodiments, the concentration of the peptide is about 0.5 mg / mL.

[0029] 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.

[0030] In some embodiments, the payload comprises an RNA molecule. In some embodiments, the RNA molecule comprises 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.

[0031] In other embodiments, the payload further comprises a protein.

[0032] In some embodiments, the nucleic acid payload encodes an immunogen. In some embodiments, the nucleic acid payload encodes hemagglutinin (HA) or ovalbumin.

[0033] In some embodiments, the ionizable lipids include lipids according to formula (I), formula (II), formula (III), formula (IV), or formula (V), or a combination thereof. In some embodiments, the ionizable lipids include at least one lipid according to formula (I). In some embodiments, the ionizable lipids include at least one lipid according to formula (II). In some embodiments, the ionizable lipids include at least one lipid according to formula (III). In some embodiments, the ionizable lipids include at least one lipid according to formula (IV). In some embodiments, the ionizable lipids include at least one lipid according to formula (V).

[0034] 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).

[0035] 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).

[0036] In some embodiments, the 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 (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 (trans-DOPE) or a combination thereof.

[0037] 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 about 20nm to about 1 μm. In some embodiments, the nanoparticles have a diameter of about 10nm to about 900nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 800nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 700nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 600nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 500nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 400nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 300nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 200nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 100nm. In some embodiments, the nanoparticles have a diameter of about 20nm to about 50nm.

[0038] 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.

[0039] In embodiments, provided herein is a method of inducing an immune response in a subject, the method comprising: administering to a subject a composition as described herein, wherein the payload is an immunogen or an encoded immunogen. In embodiments, administration comprises systemic administration or local administration, wherein local administration comprises intramuscular administration or subcutaneous administration. In embodiments, administration comprises intravenous administration. In embodiments, administration comprises administration to the brain, spinal cord, eye or lymph node of a subject. In some embodiments, the subject comprises a mammalian subject. In some embodiments, the subject comprises a human.

[0040] In some embodiments, provided herein are methods for delivering a payload to an immune cell of a subject, wherein the method comprises administering to the subject any of the compositions described herein via intravenous administration or via intramuscular administration.

[0041] In some embodiments, provided herein is a method for targeting a payload to an immune cell of a subject, the method comprising administering a composition as described herein to the subject. In embodiments, immune cells include T cells, B cells, dendritic cells (DC), T helper cells, cytotoxic T cells (CTL), natural killer cells (NK), macrophages, or a combination thereof. In other embodiments, immune cells include spleen immune cells. In some embodiments, immune cells include spleen dendritic cells.

[0042] In some embodiments, the composition targets immune cells of the 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 composition comprising an ionizable lipid and a payload without the peptide.

[0043] In various aspects, the present invention provides a method for preparing a population of lipid formulations containing a payload molecule, the method comprising: (a) mixing the payload molecule with a peptide in an aqueous solution, wherein the peptide comprises LLELLESL (SEQ ID NO: 1); (b) injecting a lipid solution comprising ionizable lipids into the aqueous solution, wherein the injecting comprises extrusion, in-line mixing, microfluidic mixing, evaporation or vortexing; and (c) producing a population of lipid formulations complexed with the payload molecule.

[0044] Also provided herein is a method for preparing a population of lipid formulations containing a payload molecule, the method comprising: (a) contacting a peptide comprising LLELLESL (SEQ ID NO: 1) with a lipid phase, wherein the lipid phase comprises an ionizable lipid, (b) contacting the components of step (a) with the payload in an aqueous solution; (c) mixing the components of step (b) by extrusion, in-line mixing, microfluidic mixing, evaporation or vortexing; and (d) producing a population of the lipid formulation complexed with the payload molecule.

[0045] 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 has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the payload molecule is a nucleic acid.

[0046] In other aspects, provided herein is a kit comprising a composition comprising 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.

[0047] Other aspects of the invention are disclosed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A-Figure 1C is a graph depicting in vivo luciferase flux localized to the spleen 4 hours after intravenous administration of lipid-peptide formulations. Figure 1A Results for different ionizable lipid formulations containing peptides are depicted. Figure 1B and Figure 1C Results for formulations with and without peptide are depicted.

[0049] Figure 2 is a graph depicting flux in the spleen following intravenous administration of lipid-peptide formulations. The graph depicts results for formulations with N / P ratios varying from 0.1 to 5.0.

[0050] Figure 3 is a graph depicting flux in muscle following intramuscular administration of lipid-peptide formulations. The graph depicts results for formulations with N / P ratios varying from 0.1 to 5.0.

[0051] Figure 4A-4Bare data showing the effect of different N / P ratios (ionizable lipids comprising charge N and nucleic acid molecules comprising charge P) on the biodistribution and delivery efficacy of formulations LP11 and LP12 after intravenous injection. Figure 4A is a graph showing the results for formulations with varying N / P ratios from 4.0 to 0.125. The bioluminescence intensity in the region of interest (ROI) was used to calculate the total flux (photons / second). Figure 4B Representative bioluminescent images showing tissue-specific delivery of formulations with N / P 2 to the spleen, but not to the liver or lung, of mice. Scale bars are radiance (p / sec / cm 2 / sr).

[0052] Figure 5 is an image showing a schematic diagram of the analysis of targeted cell populations using tdTomato reporter mice and Cre mRNA as lipoplex payload. For splenocyte analysis, splenocytes were divided into two staining groups and stained with fluorescent antibodies for CD8 (APC), CD4 (PE) and B220 (FITC) or F480 (APC), CD11b (FITC) and CD11c (PE).

[0053] Figure 6 is a graph showing specific delivery of the LP11 formulation composition containing Cre mRNA to the dendritic cell (DC) population in the spleen at administration doses of 5 μg, 10 μg, and 40 μg of Cre mRNA.

[0054] Fig. 7A is a bar graph showing tdTomato expression in different splenocyte populations after intravenous administration of lipid compositions formulated with Cre mRNA using LP11, LP12, and LP11 and LP12 formulations without the SEQ ID NO: 6 peptide (LP11-no peptide and LP12-no peptide).

[0055] Figure 7B are representative gated images showing splenic dendritic cells (MHCII+ / CD11C+ population) relative to tdTomato expression after administration of Cre mRNA formulated lipid compositions using LP11, LP12, LP11-no peptide, and LP12-no peptide.

[0056] Figure 8A-8B are data showing in vivo expression of fLuc mRNA formulated LP11 formulations or LP11 formulations without the peptide of SEQ ID NO: 6 (LP11-no peptide) at different N / P ratios. Fig. 8A Representative in vivo bioluminescence images. Scale bars are radiance (p / sec / cm 2 / sr). Figure 8Bis a graph depicting the bioluminescence intensity in the region of interest (ROI) used to calculate the total flux (photons / second).

[0057] Fig. 9 An image depicting a schematic of lipoplex uptake analysis of formulations provided herein.

[0058] Figure 10A-10B are data showing cellular uptake of mRNA complexed LP11 preparations or mRNA complexed LP11 without a SEQ ID NO: 6 peptide preparation (LP11-no peptide). Fig. 10A Representative fluorescent images of HEK293 cells at different time points after transfection are depicted (red: Cy5 mRNA, blue: DAPI nuclei; scale bar shows 10 μm). In grayscale, red is a brighter signal and blue is a darker signal. Fig. 10B is a graph depicting the quantification of cellular uptake over time after transfection.

[0059] Fig.11 An image depicting a schematic diagram of lipoplex uptake and endosomal escape analysis of formulations provided herein.

[0060] Fig.12 Depicted are representative fluorescent images showing endosomal escape of mRNA-complexed LP11 preparations or mRNA-complexed LP11 without SEQ ID NO: 6 peptide preparations (LP11-no peptide) in transfected HEK293 cells. Red: Cy5 mRNA (left two panels); Green: late endosomal marker (middle two panels); Merge: red Cy5-mRNA, green late endosomal marker and blue DAPI nuclei (right two panels); Scale bar 10 μm.

[0061] Fig.13 Image depicting a schematic diagram of the immunogenicity study design.

[0062] Figure 14A-14B is a graph showing HA antigen-specific humoral immune responses at 3 and 6 weeks. Fig.14A is a graph showing the response to the indicated mRNA doses of 0.01 mg / kg, 0.05 mg / kg or 0.25 mg / kg delivered via intramuscular (IM). Fig. 14B is a graph showing the response to the indicated mRNA doses of 0.5 mg / kg or 1 mg / kg delivered via intravenous (IV).

[0063] Figure 15A-Figure 15C These are data showing HA antigen-specific cellular immune responses. Fig.15A are exemplary images showing ELISPOT assays. Fig. 15Bis a graph showing IFN-γ secretion in response to HA peptide stimulation from splenocytes after intramuscular delivery. Fig. 15C is a graph showing IFN-γ secretion in response to HA peptide stimulation from splenocytes after intravenous delivery. DETAILED DESCRIPTION

[0064] We have developed compositions that are particularly effective in delivering payload molecules to specific tissues and / or cell types in vivo, particularly to immune system tissues and cells. The compositions comprise at least one ionizable lipid, an endosome-releasing peptide, and a payload, and provide effective and efficient delivery of payload specifically to the spleen, particularly to the dendritic cells of the spleen, after intravenous administration. After local administration (e.g., intramuscular injection), the compositions provide effective and efficient delivery of payload specifically to injection site tissues, and local immune cells. For example, antigen-specific humoral and cellular immune responses are obtained after systemic or local administration of an antigen-encoding mRNA compounded with a provided preparation.

[0065] In therapeutic applications, it is important to achieve the desired biological endpoint with the minimum amount of formulation administered, e.g., to reduce the chance of or reduce the level of associated toxicity and / or off-target effects. As demonstrated herein, the use of peptides in combination with ionizable lipids in the compositions provided results in effective and efficient delivery / uptake of payload nucleic acids, even with low levels of lipids and / or nucleic acids in the compositions. This improved efficiency allows the administration of less formulation (e.g., lower mRNA and / or lipid levels) to achieve a therapeutic effect, e.g., a specific immune response.

[0066] Exemplary payloads for delivery to immune system tissues and cells via provided lipoplex 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 lipoplex composition further comprises at least one neutral lipid.

[0067] Provided herein are compositions, methods and kits for inducing an immune response in a subject. In various aspects, a lipid composition is described, the lipid composition comprising at least one ionizable lipid comprising a charge (N), at least one peptide and a nucleic acid molecule comprising a charge (P), wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), wherein the composition has 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 may be less than about 0.1. In some embodiments, the N / P ratio is the ratio of positively charged groups (including amine (N) groups) to negatively charged groups (including phosphate (P) and peptide groups). 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 has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

[0068] In another aspect, a composition for delivering a payload to splenocytes is provided, wherein the composition comprises at least one ionizable lipid and at least one peptide comprising the sequence LLELLESL (SEQ ID NO: 1). In some embodiments, the composition for delivering a payload to splenocytes further comprises at least one neutral lipid. In some embodiments, in the composition for delivering 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 has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6). In some embodiments, the composition for delivering a payload to splenocytes further comprises at least one payload.

[0069] In particular, methods, compositions and kits for targeting a payload to immune cells, such as spleen cells, in vitro, ex vivo or in a subject are provided herein. In some embodiments of such methods,

[0070] Provided herein, inter alia, are methods, compositions, and kits for inducing an immune response in a subject.

[0071]

[0013] Provided herein, inter alia, are methods and compositions for preparing a population of lipid formulations containing a payload.

[0072] General Definition

[0073] To understand the present subject matter and to construct the appended patent claims, the following definitions are included. Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0074] Although various embodiments and aspects of the present invention are shown and described herein, it is apparent to those skilled in the art that such embodiments and aspects are provided only as examples. Without departing from the present invention, those skilled in the art will appreciate that many variations, changes and replacements are possible. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention.

[0075] The section headings used herein are for organizational purposes only and should not 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 treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0076] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those 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); Sambrook et al., MOLECULAR CLONING, ALABORATORY 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 implementation of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the present invention.

[0077] When used to refer to a numerical range, a cutoff value or a specific value, the term "about" is used to indicate that the listed value can differ from the listed value by up to 25%. Since many numerical values ​​used herein are determined by experimental methods, it should be understood by those skilled in the art that such determination can and often will vary in different experiments. Due to this inherent change, the values ​​used herein should not be considered as improper restrictions. The term "about" is used to cover changes relative to a specified value 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. Approximately 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 specified value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0078] In the description and claims herein, phrases such as "at least one of..." or "one or more of..." may appear before a list of combinations of elements or features. The term "and / or" may also appear in a list of two or more elements or features. Unless otherwise implied or explicitly contradicted by the context in which it is used, this phrase is intended to mean any element or feature listed alone, or any element or feature combined with other listed elements or features. For example, the phrases "at least one of A and B"; "one or more of A and B" and "A and / or B" are each intended to mean "A alone, B alone, or A and B together". A similar interpretation is also intended for a list including three or more items. For example, the phrases "at least one of A, B, and C"; "one or more of A, B, and C" and "A, B, and / or C" are each intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together". Furthermore, the term “based on” used above and in the claims is intended to mean “based, at least in part, on” such that non-listed features or elements are also allowable.

[0079] It should be understood that, in the case of providing a parameter range, the present invention also provides all integers and tenths thereof within the range. For example, "0.2 mg-5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc. up to 5.0 mg (including 5.0 mg).

[0080] Compounds are generally described herein using standard nomenclature. For compounds having asymmetric centers, all stereoisomers of the compound and mixtures thereof are contemplated unless otherwise indicated. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E isomers or Z isomers. When the compound exists in various tautomeric forms, the compound is intended to include all tautomeric forms. General formulas including variables (e.g., X, L1, L2, L3, Y, etc.) are used herein to describe certain compounds. Unless otherwise indicated, each variable in 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. If a portion is described as being "independently" selected from a group, each portion is selected independently of one another. Therefore, each portion may be the same or different from another portion. The number of carbon atoms in the alkyl portion may be indicated by the prefix "C x -C y” means, where x is the minimum number of carbon atoms in the moiety and y is the maximum number of carbon atoms. Thus, for example, “C1-C6 alkyl” refers to an alkyl substituent containing from 1 to 6 carbon atoms. By further example, C3-C6 cycloalkyl means a saturated hydrocarbon ring containing from 3 to 6 carbon ring atoms. A prefix attached to a multi-component substituent applies only to the first component immediately following the prefix. For illustration, the term “carbocyclylalkyl” contains two components: a carbocyclyl group and an alkyl group. Thus, for example, C3-C6 carbocyclylC1-C6 alkyl refers to a C3-C6 carbocyclyl group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0081] Unless otherwise indicated, when a linking element connects two other elements in a depicted chemical structure, the component depicted on the far left of the linking element is bound to the left element in the depicted structure, and the component depicted on the far right of the linking element is bound to the right element in the depicted structure. S -ML S ”- and M is-N(R B )S(O)-, the chemical structure is -L S -N(R B )S(O)-L S ”-.

[0082] If a connecting element in a depicted structure is a bond, then the element to the left of the connecting element is directly connected to the element to the right of the connecting element via a covalent bond. For example, if a chemical structure is depicted as -L S -ML S ' and M is chosen as a bond, the chemical structure will be -L S -L S If two or more adjacent connecting elements in the depicted structure are bonds, then the elements to the left of these connecting elements are directly connected to the elements to the right of these connecting elements via a covalent bond. For example, if a chemical structure is depicted as -L S -ML S ”-M'-L S ” - and M and L S ' is selected as a bond, the chemical structure will be -L S -M'-L S ”-. Similarly, if the chemical structure is depicted as -L S -ML S ”-M'-L S ”-, and M, L S ' and M' are bonds, then the chemical structure will be -L S -L S ”. When a chemical formula is used to describe a moiety, a dash indicates the portion of the moiety that has a free valence.

[0083] If a part is described as "optionally substituted", the part can be substituted or unsubstituted. If a part is described as optionally substituted by a non-hydrogen radical of at most a specific number, the part can be unsubstituted, or substituted by a non-hydrogen radical of at most the specific number or by a maximum number of substitutable positions on the part, whichever is smaller. Thus, for example, if a part is described as a heterocycle optionally substituted by at most three non-hydrogen radicals, any heterocycle with less than three substitutable positions will be optionally substituted by at most only as many non-hydrogen radicals as the heterocycle has substitutable positions. For example, tetrazolyl (which has only one substitutable position) will be optionally substituted by at most one non-hydrogen radical. Similarly, if amino nitrogen is described as optionally substituted by at most two non-hydrogen radicals, primary amino nitrogen will be optionally substituted by at most two non-hydrogen radicals, and secondary amino nitrogen will be optionally substituted by at most only one non-hydrogen radical.

[0084] When a moiety is substituted with oxo or thio, it means that the moiety contains a carbon atom (eg, CH2) covalently bonded to at least two hydrogens, and the two hydrogen radicals are replaced with oxo or thio to form C=O or C=S, respectively.

[0085] The term "alkenyl" means a straight or branched hydrocarbon chain containing one or more double bonds. Relative to the group substituted on the double bond carbon, each carbon-carbon double bond can have an E (cis) geometry or a Z (trans) geometry in the alkenyl portion. Examples of alkenyl radicals include, but are not limited to, vinyl, E-propenyl and Z-propenyl, isopropenyl, E-butenyl and Z-butenyl, E-isobutenyl and Z-isobutenyl, E-pentenyl and Z-pentenyl, E-hexenyl and Z-hexenyl, E, E-hexadienyl, E, Z-hexadienyl, Z, E-hexadienyl and Z, Z-hexadienyl, etc.

[0086] The term "alkenylene" refers to a divalent unsaturated hydrocarbon chain which may be linear or branched and has at least one carbon-carbon double bond. Non-limiting examples of alkenylene 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)-CH2-, -C(H)=C(H)-CH-(CH3)-, and -CH2-C(H)=C(H)-CH-(CH2CH3)-.

[0087] The term "alkyl" means a straight or branched saturated hydrocarbon chain. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl.

[0088] The term "alkylene" refers to a divalent saturated hydrocarbon chain, which may be linear or branched. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-.

[0089] The term "alkynyl" means a straight or branched hydrocarbon 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.

[0090] The term "alkynyl", alone or in combination with any other terms, refers to a straight or branched chain hydrocarbon radical having one or more triple bonds, containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 10 carbon atoms. Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl, and the like.

[0091] The term "alkoxy" refers to an alkyl ether radical, wherein the term "alkyl" is as 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.

[0092] The term "aryl", alone or in combination with any other term, refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment 6 to 15 carbon atoms (i.e. (C 6-15 ) aryl), and in another embodiment 6 to 10 carbon atoms (i.e. (C 6-10 ) aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy (e.g., methoxy), nitro, halogen (e.g., chlorine), amino, carboxylate and hydroxy. Examples of aryl radicals include, but are not limited to, phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl, etc.

[0093] The term "aralkyl", alone or in combination, means an alkyl radical as defined above wherein one of the hydrogen atoms is phenyl, benzyl, 2-phenylethyl, and the like.

[0094] The term "aralkyloxycarbonyl", used alone or in combination, means a radical of the formula -C(O)-O-aralkyl, wherein the term "aralkyl" has the meaning given above. An example of an aralkyloxycarbonyl radical is benzyloxycarbonyl.

[0095] The term "aryloxy", used alone or in combination, means a radical of the formula aryl-O-, the term "aryl" having the significance given above.

[0096] The term "alkynylene" refers to a divalent unsaturated hydrocarbon group that may be linear or branched and has at least one carbon-carbon triple bond. By way of example, representative alkynylene groups include -C≡C-, -C≡C-CH2-, -C≡C-CH2-CH2-, -CH2-C≡C-CH2-, -C≡C-CH(CH3)-, and -CH2-C≡C-CH(CH2CH3)-.

[0097] The term "alkanoyl," used alone or in combination, means an acyl radical derived from an alkanoic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, and the like.

[0098] The term "aryloxyalkanoyl" is intended to indicate an acyl radical of the formula aryl-O-alkanoyl in which aryl and alkanoyl have the meanings given above.

[0099] The term "aralkanoyl" means an acyl radical derived from an aryl-substituted alkanecarboxylic acid, such as phenylacetyl, 3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyroyl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-phenylbutyroyl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, and the like.

[0100] The term "aroyl" means an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, optionally substituted benzoic acid or naphthoic acid, such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4-(benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3-(benzyloxycarbonyl)-2-naphthoyl, etc.

[0101] The term "aminocarbonyl", used alone or in combination, means an amino-substituted carbonyl (carbamoyl) derived from an amino-substituted carboxylic acid, wherein the amino group can be a primary amino group continuous substituent, a secondary amino group continuous substituent, or a tertiary amino group continuous substituent selected from hydrogen radicals, alkyl radicals, aryl radicals, aralkyl radicals, cycloalkyl radicals, cycloalkylalkyl radicals, and the like.

[0102] The term "aminoalkanoyl" means an acyl radical derived from an amino-substituted alkanecarboxylic acid, wherein the amino group may be a primary, secondary or tertiary amino group containing a substituent selected from the group consisting of a hydrogen radical, a cycloalkyl radical, a cycloalkylalkyl radical, and the like, examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.

[0103] The term "carbocycle" or "carbocyclic" or "carbocyclyl" refers 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 zero heteroatom ring atoms. "Ring atoms" or "ring members" are atoms that are bound together to form a ring. A carbocyclyl can be, but is not limited to, a single ring, two fused rings, or a bridged ring or a spirocycle. Substituted carbocyclyls can have cis or trans geometry. Representative examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, decahydronaphthyl and norpinyl.Carbocyclyl groups can be attached to the parent molecular moiety by any replaceable carbon ring atom. In the case where the carbocyclyl group is a divalent part connecting two other elements in the depicted chemical structure, the carbocyclyl group can be attached to the two other elements by any two replaceable ring atoms. Similarly, in the case where the carbocyclyl group is a trivalent part connecting three other elements in the depicted chemical structure, the carbocyclyl group can be attached to the three other elements by any three replaceable ring atoms respectively. Carbocycle can be attached to any bridge ring carbon atom producing a stable structure. In one embodiment, the carbocycle has 5 to 7 carbons.

[0104] The term "cycloalkyl" refers to a saturated carbocyclyl group containing zero heteroatom ring members. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl, and norpinanyl.

[0105] The term "cycloalkyl", used alone or in combination, means an alkyl radical containing about 3 to about 8 carbon atoms and being cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0106] The term "cycloalkylalkyl" means an alkyl radical as defined above substituted with a cycloalkyl radical containing from about 3 to about 8, and in one embodiment from about 3 to about 6, carbon atoms.

[0107] The term "cycloalkylcarbonyl" means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid, such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, etc., or an acyl group derived from a benzo-fused monocyclic cycloalkanecarboxylic acid optionally substituted with, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2-naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0108] The term "cycloalkylalkoxycarbonyl" means an acyl radical derived from a cycloalkylalkoxycarboxylic acid of formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.

[0109] The term "carbocyclylalkyl" refers to a carbocyclyl group attached to the parent molecular moiety through an alkylene group. For example, C3-C6carbocyclylC1-C6alkyl refers to a C3-C6carbocyclyl group attached to the parent molecular moiety through a C1-C6alkylene group.

[0110] The term "cycloalkenyl" refers to a non-aromatic, partially unsaturated carbocyclyl moiety having zero heteroatom ring members. Representative examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, and octahydronaphthyl.

[0111] The prefix "halo" means that the substituent to which the prefix is ​​attached is substituted by 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 substituted by 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 appreciated that if a substituent is substituted by more than one halogen radical, those halogen radicals may be the same or different (unless otherwise stated).

[0112] The term "heterocycle" or "heterocyclic" or "heterocyclyl" refers to a saturated (e.g., "heterocycloalkyl"), partially unsaturated (e.g., "heterocycloalkenyl" or "heterocycloalkynyl") or fully unsaturated (e.g., "heteroaryl") ring system, wherein 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 heterocycle can be, but is not limited to, a single ring, two fused rings, or a bridged ring or a spirocycle. The heterocyclic group can be connected to the parent molecular part via any substitutable carbon atom or nitrogen atom in the group. In the case where the heterocyclic group is a divalent part connecting two other elements in the depicted chemical structure, the heterocyclic group can be attached to the two other elements by any two substitutable ring atoms. Similarly, in the case where the heterocyclic group is a trivalent part connecting three other elements in the depicted chemical structure, the heterocyclic group can be attached to the three other elements by any three substitutable ring atoms, respectively.

[0113] In the compounds of the present invention, "Het" represents a heterocycle containing 4 to 12 carbon atoms, wherein at least one nitrogen atom is present in the ring. The heterocyclic radical 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, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiol, oxathiol, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiadiazolyl, oxathiazolyl, oxadiazolyl (including 1,2, 3-oxadiazolyl, 1,2,4-oxadiazolyl (also known as "oxadiazolyl"), 1,2,5-oxadiazolyl (also known as "furoxyl") 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, dioxazolyl azinyl (including pyridazinyl (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- The invention also includes oxazinyl, 1,3,6-oxazinyl (also known as "pentyloxazolone", 1,2,6-oxazinyl and 1,4-oxazinyl), isoalloxazinyl (including o-isoalloxazinyl and p-isoalloxazinyl), oxazolidinyl, isoxazolidinyl, oxathiazinyl (including 1,2,5-oxathiazinyl or 1,2,6-oxathiazinyl), oxadiazinyl (including 1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, azepinyl and diazepinyl.

[0114] The heterocyclic group can also be, but is not limited to, a bicyclic ring containing two fused rings, such as, for example, naphthyridinyl (including [1,8] naphthyridinyl and [1,6] naphthyridinyl), thiazolylpyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyridoindenyl, pyranopyrrolyl, 4H-quinolizinyl, purinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]-pyridinyl and pyrido[4,3-b]-pyridinyl), pyridopyrimidine and pteridinyl. Other non-limiting examples of fused-ring heterocycles include benzo-fused heterocyclic groups such as indolyl, isoindolyl, indoleninyl (also known as "pseudoindolyl"), isoindazolyl (also known as "benzopyrazolyl" or indazolyl), benzopyridinyl (including quinolinyl (also known as "1-benzopyridinyl") and isoquinolinyl (also known as "2-benzopyridinyl")), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including quinolinyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including quinolinyl, phthalazinyl, quinoxalinyl, benzodiazinyl), benzophenone, ... quinazolinyl (also known as "1,2-benzodiazinyl" and quinazolinyl (also known as "1,3-benzodiazinyl")), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, 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-benzoisoxazinyl and 1,4-benzoisoxazinyl) and tetrahydroisoquinolinyl.

[0115] The heterocyclic group may also be, but is not limited to, a spirocyclic system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decyl. The heterocyclic group may contain one or more sulfur atoms as ring members; and in some cases, the sulfur atom is oxidized to SO or SO2. The nitrogen heteroatom in the heterocyclic group may be quaternized or not, and may be oxidized or not to N-oxide. In addition, the nitrogen heteroatom may or may not be N-protected.

[0116] The heterocyclic or carbocyclic ring may be further substituted. Unless otherwise indicated, the term "substituted" refers to substitution by independently replacing one, two, three or more hydrogen atoms with substituents, including but not limited to -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO2, -N3, -CN, -NH2, protected amino, oxo, thio, -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-C12 -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, -NHCO2aryl, -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 -aryl, -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 -cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl or methylthiomethyl. It should be understood that aryl, heteroaryl, alkyl, etc. may be further substituted.

[0117] The term "N-protecting group" or "N-protected" refers to those groups that are capable of protecting an amino group from undesired reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3rd ed., John Wiley & Sons, NY (1999). Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl or 4-nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfinyl groups, Such as phenylsulfinyl (phenyl-S-), triphenylmethylsulfinyl (trityl-S-); sulfinyl groups such as p-methylphenylsulfinyl (p-methylphenyl-S(O)-), tert-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, Oxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethyloxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloro-ethoxy- The N-protecting groups include phenyl, phenyl, tert-butyl ...

[0118] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0119] The term "biologically active agent / bioactive agents" and the like generally refers 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 may be, but need not be, an endogenous molecule (e.g., a molecule such as a protein or nucleic acid that is normally present in a target cell). The biological effect may be, but is not limited to, a biological effect that stimulates or causes an immunoreactive response; a biological effect that affects a biological process in a cell, tissue or organism (e.g., an animal); a biological effect that imparts a biological process in a pathogen or parasite; a biological effect that produces or causes a detectable signal; a biological effect that modulates the expression of a protein or polypeptide; a biological effect that terminates or inhibits the expression of a protein or polypeptide; or a biological effect that causes or enhances the expression of a protein or polypeptide. The biologically active composition, complex, compound or molecule may be used in research, therapeutic, prophylactic and diagnostic methods and compositions and is generally used for etiology.

[0120] As used herein, the term "transfection enhancer / transfection enhancing agent" refers to a compound that, when added to a transfection agent, increases transfection efficiency (i.e., increases the percentage of cells transfected), increases the expression level of the transfection agent, or reduces the amount of payload (e.g., nucleic acid or protein) required to obtain a biological response, or any combination of the above enhancements. In some embodiments, transfection enhancers also facilitate the delivery of molecules that help to downregulate expression, such as siRNA, LNA, etc. In some embodiments, transfection enhancers are peptides, including, for example, cell surface ligands, fusogens, and / or nuclear localization agents (such as nuclear receptor ligand peptides). Transfection enhancers include, for example, the peptides and polypeptides described in Table 1 of U.S. Patent No. 10,538,784, the contents of which are hereby incorporated by reference in their entirety.

[0121] As used herein, the term "nucleic acid binding moiety" refers to a compound or molecule that is capable of binding to a nucleic acid. In some embodiments, the binding molecule is capable of non-covalent binding to a nucleic acid, while in other embodiments, the binding molecule is covalently linked to a transfection enhancer, a cell surface ligand, a nuclear localization sequence, and / or a fusogenic agent. The binding molecule may include, but is not limited to, spermine, spermine derivatives, spermidine, histones or fragments thereof, protamine or fragments thereof, HMG protein 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.

[0122] As used herein, the term "polycationic nucleic acid binding moiety" refers to a portion containing multiple positive charges at physiological pH that allows the portion to bind negatively charged nucleic acids. The polycationic nucleic acid binding moiety can be connected to, for example, a transfection enhancer, such as a cell surface ligand, a fusogenic agent, and / or a nuclear localization peptide. The connection can be covalent. Suitable polycationic nucleic acid binding moieties include polyamines and polycyclic peptides containing, for example, multiple lysine, arginine, ornithine or histidine residues (such as about 8-20 such residues). Non-limiting examples are cationic peptides of repeat sequences of lysine or arginine, for example, sequences with 8-20 lysine residues or 8-20 arginine residues.

[0123] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein in embodiments the polymer may be combined with a moiety that does not consist of amino acids. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding amino acid occurring in nature, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. "Fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single part.

[0124] As used herein, the term "lytic agent" or "endosome releasing agent" refers 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, including 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 their derivatives. The term "lytic peptide" refers to any chemical grouping that permeates the membrane so that the structural organization and integrity of the membrane are lost. Due to the presence of a 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 in, for example, Pei and Buyanova, Bioconjugate Chem, 30: 273-283 (2009) and Juliano, Nucleic Acid Therapeutics, 28: 166-177 (2018).

[0125] The term "surface ligand" or "cell surface ligand" refers to a chemical compound or structure that will bind to a surface receptor of a cell. As used herein, the term "cell surface receptor" refers to a specific grouping of chemicals on the surface of a cell to which a ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one type of cell but not in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). Receptors promote internalization of ligands and attachment molecules. Cell surface receptors include, but are not limited to, folate receptors, biotin receptors, lipoic acid receptors, low-density lipoprotein receptors, asialoglycoprotein receptors, insulin-like growth factor type II / cation-independent mannose-6-phosphate receptors, calcitonin gene-related peptide receptors, insulin-like growth factor I receptors, nicotinic acetylcholine receptors, hepatocyte growth factor receptors, endothelin receptors, bile acid receptors, bone morphogenetic protein receptors, cartilage inducing factor receptors, or glycosylphosphatidylinositol (GPI) anchored proteins (e.g., β-adrenergic receptors, T cell activation protein, Thy-1 protein, GPI anchored 5' nucleotidase). These are non-limiting examples.

[0126] "Receptor" is a molecule that a ligand specifically binds and has a relatively high affinity. Receptors are usually proteins or glycoproteins, but can also be glycolipids, lipopolysaccharides, glycosaminoglycans or polysaccharide coatings. For the purposes of this disclosure, the epitope to which an antibody or fragment thereof binds is interpreted as a receptor because the antigen: antibody complex is endocytosed. In addition, surface ligands include any substance that can enter a cell by cellular phagocytosis (e.g., endocytosis, fluid uptake, pinocytosis). As used herein, the term "ligand" refers to a compound or structure that will bind to a receptor. This includes, but is not limited to, ligands such as asialo-seromucoids, asialoglycoproteins, lipoic acid, biotin, apolipoprotein E sequences, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, cell adhesion peptides containing RGD, etc. The ligand can also be a plant virus movement protein or a peptide derived from such a protein. Suitable peptides and proteins are described in, for example, U.S. Patent No. 10,538,784, the contents of which are hereby incorporated by reference in their entirety. One skilled in the art will readily recognize that the selected ligand will depend on which receptor is bound. Because different types of cells have different receptors, this provides a method of targeting a payload (such as a polypeptide or nucleic acid molecule) to a specific cell type, depending on which cell surface ligand is used. Therefore, the use of cell surface ligands can depend on the targeted cell type.

[0127] Composition

[0128] Provided herein are lipid compositions 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 has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 1. In some embodiments, the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-24.

[0129] Exemplary payloads for delivery to tissues and cells via provided lipoplex 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.

[0130] In some embodiments of the lipid composition, a payload can be selected, such as one or more nucleic acids (e.g., mRNA, siRNA, sgRNA), lipids, and their amounts to provide a specific N / P ratio. The N / P ratio of a composition refers to the molar ratio of the number of phosphate groups in the ionizable (at physiological pH) nitrogen atoms in one or more lipids to the number of phosphate groups in the nucleic acid (e.g., RNA). For example, Schoenmaker et al. (International Journal of Pharmaceutics; 601 (2021), which is incorporated herein by reference in its entirety) discuss RNA-lipid nanoparticle N / P ratios (e.g., mRNA and siRNA payloads), such as in Table 1 and page 4.

[0131] In certain embodiments, a lower N / P ratio is preferred. Provided herein are lipid compositions comprising at least one ionizable lipid having a charge (N), at least one peptide, and a nucleic acid molecule comprising a charge (P), wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), wherein the composition has 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. Provided herein are lipid compositions comprising at least one ionizable lipid having a charge (N), at least one neutral lipid, at least one peptide, and a nucleic acid molecule comprising a charge (P), wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), wherein the composition has 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 0.01 to 0.10. In other examples, the N / P ratio is 0.01 to 0.20. In other examples, the N / P ratio is 0.01 to 0.25. In other examples, the N / P ratio is 0.01 to 0.33. In other examples, the N / P ratio is 0.01 to 0.5. In other examples, the N / P ratio is 0.01 to 1. In other examples, the N / P ratio is 0.05 to 0.1. In other examples, the N / P ratio is 0.05 to 0.125. In other examples, the N / P ratio is 0.5 to 0.15. In other examples, the N / P ratio is 0.05 to 0.167. In other examples, the N / P ratio is 0.05 to 0.20. In other examples, the N / P ratio is 0.05 to 0.25. In other examples, the N / P ratio is 0.05 to 0.33. In other examples, the N / P ratio is 0.05 to 0.5. In other examples, the N / P ratio is 0.05 to 1.0. In some embodiments, the N / P ratio may 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.

[0132] 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 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 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.

[0133] 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 about 5 to about 10. In other embodiments, the N / P ratio is about 5 to about 20. In other embodiments, the N / P ratio can be about 10 to about 20, about 10 to about 30, about 15 to about 30, about 15 to about 40, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 50, about 30 to about 40, or about 35 to about 50.

[0134] The ionizable lipid and peptide compositions provided herein encompass complexes in the form of lipid nanoparticles, liposomes (e.g., lipid vesicles) and liposome complexes. As used herein, the term "liposome" encompasses any compartment encapsulated by a lipid bilayer. The term liposome includes a unilamellar capsule, which is composed of a single lipid bilayer and generally has a diameter in the range of about 20nm to about 400nm. Liposomes can also be multi-chambers with a diameter in the range of about 1 μm to about 10 μm. Multi-chamber liposomes can be composed of several (from two to hundreds) unilamellar capsules, forming one inside another, with a gradually reduced size, producing a multi-chamber structure of concentric phospholipid spheres separated by a water layer. Alternatively, multi-chamber liposomes can be composed of many smaller non-concentric lipid spheres within a large liposome. In embodiments, liposomes include multi-chamber capsules (MLVs), large unilamellar capsules (LUVs) and small unilamellar capsules (SUVs). In some embodiments, the composition comprises liposomes containing any suitable ionizable lipids and neutral lipids and peptides provided herein.

[0135] In some embodiments, the composition comprises lipid nanoparticles (LNP). The size of the LNP composition is generally micron-sized or small, and may include a lipid bilayer. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 1 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 400 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 100 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 50 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 400 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 150 μm.

[0136] The characteristic of lipid nanoparticle (for example, LNP without payload or LNP containing payload) can depend on its component.For example, the lipid nanoparticle comprising cholesterol as structural lipid can have the characteristic different from the lipid nanoparticle comprising different structural lipid.Similarly, the characteristic of lipid nanoparticle (for example, LNP without payload or LNP containing payload) can depend on the absolute amount or relative amount of its component.For example, the lipid nanoparticle comprising the phospholipid of higher mole fraction can have the characteristic different from the lipid nanoparticle comprising the phospholipid of lower mole fraction.Characteristic can also change according to the method and condition of preparing nanoparticle composition.

[0137] Lipid nanoparticles can be characterized by a variety of methods. For example, a microscope (e.g., a transmission electron microscope or a scanning electron microscope) can be used to examine the morphology and size distribution of the nanoparticle composition. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure the various characteristics of the nanoparticle composition, such as particle size, polydispersity index and zeta potential.

[0138] The lipid complex compositions provided herein can be relatively uniform. The polydispersity index can be used to represent the uniformity of the nanoparticle composition, such as the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally represents a narrow particle size distribution. The 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., LNPs without a payload or LNPs containing a payload) can be about 0.10 to about 0.20.

[0139] The zeta potential of lipid nanoparticles (e.g., LNPs without or containing a useful load) can be used to represent the electrokinetic potential of a composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Lipid nanoparticles with relatively low charge (positive or negative charge) are typically desirable because more highly charged materials may interact undesirably with cells, tissues, and other elements in vivo. In some embodiments, the zeta potential of the 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 to about +15 mV, or about +5 mV to about +10 mV.

[0140] In some embodiments, the payload of the lipoplex 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 2 mRNA molecules, at least 3 mRNA molecules, at least four mRNA molecules, or at least 5 mRNA molecules). In some embodiments, the payload includes at least one sgRNA. In other embodiments, the payload of the lipoplex composition molecule includes sgRNA molecules and mRNA molecules.

[0141] In some embodiments, the lipid composition payload includes a gene editing reagent (or a 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, a MegaTal, a Cre recombinase, a Hin recombinase or a Flp recombinase. In some embodiments, the RNA molecule includes sgRNA, crRNA and / or tracrRNA. Therefore, in some embodiments, the lipid complex composition payload includes RNP and sgRNA. In some embodiments, RNP may include a Cas protein and sgRNA, crRNA or tracrRNA.

[0142] In other embodiments, the RNA may encode a gene editing protein (e.g., RNA encoding a ZFN, TALEN, Cas protein, Cre recombinase, etc.). Thus, in some embodiments, the lipoplex composition payload includes RNA encoding a gene editing protein and sgRNA. In some embodiments, the lipoplex composition payload may include RNA encoding a Cas protein and sgRNA, crRNA, or tracrRNA.

[0143] In some embodiments, the nucleic acid payload of the lipoplex composition is a single-stranded molecule. In some embodiments, the payload may include donor DNA. In other embodiments, the DNA payload may be plasmid DNA or linear DNA. In some examples, the payload may be RNP and include Cas protein and sgRNA, crRNA or tracrRNA.

[0144] In certain embodiments, the gene editing payload induces single-strand or double-strand 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 a 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 a 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 a CRISPR-Cas system is discussed in Ran et al. (2013) Nat Protoc. 2013 November; 8(11): 2281-2308, the entire contents of which are incorporated herein by reference. Embodiments involving repair templates are not limited to those comprising a CRISPR-Cas system.

[0145] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database with accession number Q99ZW2 and in the NCBI database with accession number Q99ZW2.1. UniProt database accession numbers A0A0G4DEU5 and CDJ55032 provide another example of a Cas9 protein amino acid sequence. Another non-limiting example is the Streptococcus thermophilus Cas9 protein, the amino acid sequence of which can be found in the UniProt database with accession number Q03JI6.1. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In certain embodiments, the CRISPR enzyme is Cas9, and may be Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae. In various embodiments, the CRISPR enzyme guides the cutting of one or both chains at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme guides the cleavage of one or two chains 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 is mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or two chains of the target polynucleotide containing the target sequence. For example, the aspartic acid that occurs in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes is replaced by alanine to convert Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A. In aspects of the present invention, nickases can be used for genome editing via homologous recombination.

[0146] In certain embodiments, the payload may include a Cas9 nickase used in combination with a guide sequence (e.g., two guide sequences) that targets the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ.

[0147] In yet other examples, the payload of the lipid composition includes an RNA molecule, and the RNA molecule includes sgRNA, crRNA, tracrRNA, or a combination thereof.

[0148] 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 an ovalbumin or a fragment thereof. In examples, the lipid composition delivers a payload that induces an immune response in a subject to a protein of the lipid composition or a nucleic acid encoding a protein.

[0149] Exemplary transfection enhancing peptides for 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 portion. In some embodiments, the peptide comprises 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 peptide comprises one or more PSYYRYD (SEQ ID NO: 25) or PSYYRGD (SEQ ID NO: 26) sequences and optionally comprises a polycationic nucleic acid binding portion.

[0150] As described herein, suitable polycationic nucleic acid binding moieties include, but are not limited to, polyamines and polycyclic peptides containing, for example, polyarginine, polylysine, polyhistidine and / or polyornithine sequences of, for example, about 8 to about 20 residues in length.

[0151] The length of the peptide used in the compositions provided herein is at least 10 amino acids. In some embodiments, the length of the peptide is 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 certain embodiments, the length of the peptide is 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 some embodiments, the length of the peptide is 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.

[0152] In some examples, the peptide has at least 80% sequence identity with SEQ ID NO:6 (GLFEALLELLESLWELLLEA). In other examples, the peptide includes 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 to SEQ ID NO: 6. In other examples, the peptide has at least 99% sequence identity to SEQ ID NO: 6. In other examples, the peptide has 100% sequence identity to SEQ ID NO: 6.

[0153] A "polypeptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, wherein the remaining amino acid sequence is generally identical to the corresponding positions in the naturally occurring sequence. Fragments are generally at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long, or at least 70 amino acids long.

[0154] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, wherein the polynucleotide or polypeptide sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) to achieve optimal alignment of the two sequences. In an embodiment, the percentage is calculated as follows: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the percentage of sequence identity.

[0155] The term "identical" or percentage "identity" in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when comparing and comparing the maximum correspondence on a comparison window or a specified region (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity on a specified region of, for example, a complete polypeptide sequence or its individual domains), as measured using a sequence comparison algorithm or by manual comparison and visual inspection. In an embodiment, two sequences are 100% identical. In an embodiment, two sequences are 100% identical in the total length of one of the sequences (e.g., in the case where the sequences have different lengths, the shorter of the two sequences). In an embodiment, identity can refer to the complementary sequence of a test sequence. In embodiments, identity exists 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 exists 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.

[0156] Exemplary peptides that can be used in the formulations provided herein include, but are not limited to, the peptides of Table 1.

[0157] Table 1: Peptide sequences

[0158] SEQ ID NO: sequence 6 GLFEALLELLESLWELLLEA 7 GLFEALLELLESLYELLLEA 8 GLFEALLELLESLWELLLEAWYG 9 GLFEALLELLESLWELLLEAKK 10 GLFEALLELLESLWELLLEAKKKK 11 GLFEALLELLESLWELLLEAKKKKKK 12 GLFEALLELLESLWELLLEAKKKKKKKK 13 GLFEALLELLESLWELLLEAEE 14 GLFEALLELLESLWELLLEAEEEE 15 GLFEALLELLESLWELLLEAEEEEEE 16 GLFEALLELLESLWELLLEAEEEEEEEE 17 GLFEALLELLESLWELLLEARR 18 GLFEALLELLESLWELLLEARRRR 19 GLFEALLELLESLWELLLEARRRRRR 20 GLFEALLELLESLWELLLEARRRRRRRR 21 GLFEALLELLESLWELLL 22 GLLEELLELLEELWEELLEG 23 GLFEALLELLESLWELLLEAGSGGSGSGSRRRRRRRRRR 24 GLFEALLELLESLWELLLEAGSSSSGSSSGSSSRRRRRRRRRR

[0159] In some embodiments, any of the peptides of SEQ ID NO: 1-22 may also include an arginine residue or an additional arginine residue (e.g., R2, R4, R6, R8, R12) at the N or C terminus. In other embodiments, any of the peptides of SEQ ID NO: 1-22 may also include a lysine residue or an 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 NO: 1-22 may include multiple 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 NO: 1-22 may include multiple ornithine residues at the N or C terminus. In certain embodiments, the sequences of SEQ ID NO: 1-22 are linked to multiple arginine, lysine, histidine, or ornithine residues via a spacer or linker molecule.

[0160] 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 with any one of SEQ ID NOs: 6-24, the formulation may further comprise an additional transfection enhancer, such as a cell surface ligand peptide and / or a nuclear localization agent, such as a nuclear receptor ligand peptide. Examples of such transfection enhancers include, but are not limited to, reovirus-associated gene fusion peptides, insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell targeting antibodies, lactoferrin, fibronectin, adenovirus penton bases, knobs, hexon proteins, vesicular stomatitis virus glycoproteins, victorin 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 Sex group proteins and invasin proteins, internalizing protein proteins, endotoxins, diphtheria toxins, Shigella toxins, melittin, magainin, gramicidin, cecropin, defensins, antimicrobial peptides, horseshoe crab, sulfonin, indolicidin, bovine antimicrobial peptides, drosophilin, bee antimicrobial peptides, cathepsin inhibitors, bactericidal-permeable-increasing proteins, nisin, toad aglycone and fragments thereof, as well as those disclosed in U.S. Application Publication No. 2018 / 0340188 (which is hereby incorporated by reference in its entirety).

[0161] Exemplary transfection enhancing peptides useful in the formulations provided herein include peptides comprising one or more sequences of PSYYRYD (SEQ ID NO: 25) and / or PSYYRGD (SEQ ID NO: 26), including but not limited to the exemplary peptides of Table 2.

[0162] Table 2

[0163] SEQ ID NO: sequence 27 AARSPSYYRGDYGPYYAMDYD RRRRRRRRRRRR 28 AARSPSYYRGDYGPYYAMDYD 29 AARSPSYYRGDAGPYYAMDYDRRRRRRRRRRRR 30 AARSPSYYRGDAGPYYAMDYD 31 AARSPSYYRYDYGPYYAMDYD RRRRRRRRRRRR 32 AARSPSYYRYDYGPYYAMDYDR 33 PSYYRGDGAPSYYRGDGAPSYYRGDGARRRRRRRRRRRR 34 PSYYRGDGAPSYYRGDGAPSYYRGDGA 35 PSYYRYDYGPSYYRYDYGPSYYRYDYGRRRRRRRRRRRRR 36 PSYYRYDYGPSYYRYDYGPSYYRYDYG

[0164] 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 SEQ ID NOs: 27-36 peptides may also include additional arginine residues at the N or C terminus (e.g., R2, R4, R6, R8, R10, R12, R14, R16, R18, R20). In other embodiments, any one of SEQ ID NOs: 28, 30, 32, 34, and 36 may also include lysine residues at the N or C terminus (e.g., K2, K4, K6, K8, K10, K12, K14, K16, K18, K20). In other embodiments, any one of the SEQ ID NOs: 27-36 peptides may include multiple histidine residues at the N or C terminus (e.g., H2, H4, H6, H8, H12). In other embodiments, any of the SEQ ID NOs: 27-36 peptides may comprise multiple ornithine residues at the N or C terminus. In certain embodiments, the SEQ ID NOs: 27-36 sequences are linked to multiple arginine, lysine, histidine or ornithine residues via a spacer or linker molecule.

[0165] In some embodiments, the lipid compositions provided include a peptide 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 lipid compositions provided include a peptide having at least 80% sequence identity to SEQ ID NO: 6 and a peptide having at least 80% sequence identity to SEQ ID NO: 27. In other embodiments, the lipid compositions include a peptide having at least 80% sequence identity to SEQ ID NO: 6 and a peptide having at least 80% sequence identity to SEQ ID NO: 33. In some embodiments, the lipid compositions include a peptide selected from the group consisting of peptides of SEQ ID NOs: 6-24 and a peptide selected from the group consisting of peptides of SEQ ID NOs: 27-36.

[0166] In some embodiments, the endosomal releasing peptides and / or cell surface ligand peptides used in provided compositions can be linked to a glycosylphosphatidylinositol (GPI) anchor peptide, such as FTLTGLLGTLVTMGLLT (SEQ ID NO: 37).

[0167] In some embodiments, the peptides described herein are directly attached to the nucleic acid binding molecule by covalent bonding or are connected to the binding molecule via a spacer. As used herein, the terms "spacer" or "linker" used interchangeably herein refer to a chemical structure that connects two molecules to each other. In some embodiments, the spacer is attached to each molecule on a different part of the spacer molecule. In other embodiments, the spacer is a hydrophilic moiety and comprises about 6 to 30 carbon atoms. In other embodiments, the spacer comprises a polyether, such as -CH2-O-(CH2-CH2-O-) i In other embodiments, the spacer comprises a hydrophilic polymer, such as [(gly) i (ser) j ] k In these formulas, i is in the range of 1 to 6, j is in the range of 1 to 6, and k is in the range of 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.

[0168] The length of the peptide used in the compositions provided herein is at least 10 amino acids. In some embodiments, the length of the peptide is 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 certain embodiments, the length of the peptide is 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 some embodiments, the length of the peptide is 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.

[0169] In some embodiments, the lipid compositions provided include peptides, wherein the concentration of the peptides is from about 0.001 mg / mL to about 0.5 mg / mL. In embodiments, the concentration of the peptides is from about 0.05 mg / mL to about 0.5 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.001 mg / mL to about 0.05 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.001 mg / mL to about 0.1 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.01 mg / mL to about 0.5 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.01 mg / mL to about 0.4 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.01 mg / mL to about 0.3 mg / mL. In examples, the lipid compositions include peptides, wherein the concentration of the peptides is from about 0.01 mg / mL to about 0.2 mg / mL. In an example, the lipid composition comprises a peptide, wherein the concentration of the peptide is about 0.01 mg / mL to about 0.1 mg / mL. In an embodiment, the concentration of the peptide is about 0.05 mg / mL to about 0.1 mg / mL. In an embodiment, the concentration of the peptide is about 0.05 mg / mL to about 0.2 mg / mL. In an embodiment, the concentration of the peptide is about 0.05 mg / mL to about 0.3 mg / mL. In an embodiment, the concentration of the peptide is about 0.05 mg / mL to about 0.4 mg / mL. In an embodiment, the concentration of the peptide is about 0.1 mg / mL to about 0.5 mg / mL. In an embodiment, the concentration of the peptide is about 0.1 mg / mL to about 0.4 mg / mL. In an embodiment, the concentration of the peptide is about 0.1 mg / mL to about 0.3 mg / mL. In an embodiment, the concentration of the peptide is about 0.1 mg / mL to about 0.2 mg / mL.

[0170] 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 medium containing the lipid. The functional group can be basic, such as an amino functional group, or can be acidic, such as a carboxylic acid moiety. Those skilled in the art will appreciate that other ionizable functional groups can also be used. The ionizable lipid can contain both a basic moiety and an acidic moiety. Advantageously, the ionizable lipid carries an overall positive charge at physiological pH.

[0171] Ionizable lipids as described herein refer to lipids having at least one protonatable group or deprotonatable group, such that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4), and is neutral at a second pH, preferably at or higher than physiological pH. Preferably, when incorporated into a lipid composition (e.g., liposomes, lipid nanoparticles or other lipid complexes), the ionizable lipids provided herein have a pKa of a protonatable group in the range of about 4 to about 11, e.g., about 4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and 6.9. Ionizable lipids include, for example, amine-containing lipids that can be easily protonated.

[0172] The ionizable lipid may be selected, for example, from the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine. )-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, L-spermine-5-carboxy-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-dilysyl)-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)-oleylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, 1,4-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-myristylamino]-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)-myristylamino)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-carboxy-amidoethylenetrimethylammonium iodide, 1,4-bis[(3-myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl formate iodide, cholesteryl-3β-carboxyamido Ethyleneamine, cholesterol-3β-oxysuccinylaminoethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonium)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornithineglycyl-N-(1-heptadecylodecyl)glycinamide, N, 2 ,N 5 -bis(3-aminopropyl)-L-ornithineglycyl-N-(1-heptadecanyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dioctadecyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-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-ornithine]-NN-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-ornithine-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N5 -bis(3-aminopropyl)-L-ornithine]-N,N-dioleyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-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-ornithine-NN-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-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-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-ornithine-NN-dipalmitoyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dimyristyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-glutamine, N 2 -[N2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-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-ornithine]-N,N-dilauroyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauroyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauroyl-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-ornithine-NN-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-dipalmitylpropionamide, 3-[N',N"-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propionamide [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]methylammonium bromide ethyl)] methylammonium bromide, 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-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)->4-(3-amino-propylamino)-4-carbamoylbutylcarbamoyl cholesterol ester, (3-amino- Spermidine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide tetrakis trifluoroacetate, Spermidine-5-carboxyglycine (N'-stearyl-N'-antioleyl) amide tetrakis trifluoroacetate, Agmatine carboxycholesterol acetate, Spermidine-5-carboxy-β-alanine cholesteryl tetrakis trifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesteryl bis trifluoroacetate, 2,4-diaminobutyryl β-alanine cholesteryl bis trifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl tris trifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl] aminocarboxy cholesterol ester, stearyl carnitine ester, palmityl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester chloride, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester chloride, palmityl palmitoyl carnitine ester chloride, myristyl myristoyl carnitine ester chloride, L-myristyl myristoyl carnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis-(oleyloxyethyl)-piperazinium bromide, N-(3 -aminopropyl)-N,N'-bis-(palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis-(myristyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristyloxyethyl)-piperazinium bromide, 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)-palmitylamino)-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), heptathriacontac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (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)-octadec-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)-octadec-9,12-dien-1-yloxy]propan-1-amine12-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)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)).

[0173] In some embodiments, the ionizable lipids described in U.S. Pat. No. 7,173,154, U.S. Pat. No. 9,856,496, and U.S. Patent Publication No. US2019 / 0060482 (which references are incorporated by reference in their entirety) are contemplated for use in the compositions and methods of the present invention.

[0174] In some preferred but non-limiting embodiments, the lipid composition may comprise at least a first ionizable lipid and at least a first neutral lipid, wherein the lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein the ionizable lipid has a structure of formula (I):

[0175] and salts thereof; wherein:

[0176] R1 and R2 are independently an alkyl, alkenyl or alkynyl group having 8 to 30 carbon atoms;

[0177] An alkyl, alkenyl or alkynyl group having 8 to 30 carbon atoms and optionally substituted with one or more of an alcohol, amino alcohol, amine, amide, ether, polyether, ester, thiol, alkylthio or carbamoyl group or wherein R1 is -(CH2) q -N(R6) t R7R8;

[0178] R3 and R4 are independently hydrogen or an alkyl, alkenyl or alkynyl group having 8 to 30 carbon atoms and are optionally substituted with one or more of an alcohol, amino alcohol, amine, amide, ether, polyether, ester, thiol, alkylthio or carbamoyl group;

[0179] R5-R8 are independently hydrogen or an alkyl, alkenyl or alkynyl group;

[0180] R9 is hydrogen or an alkyl, alkenyl or alkynyl group, a carbohydrate or a peptide;

[0181] r, s, and t are 1 or 0 to indicate the presence or absence of the indicated R group, when any one of r, s, or t is 1, the nitrogen to which the indicated R group is attached is positively charged, and wherein at least one of r, s, or t is 1;

[0182] q is an integer from 1 to 6, inclusive;

[0183] X v- is an anion, where v is the valence of the anion and A is the number of anions;

[0184] L is a divalent organic group capable of covalently linking two nitrogens selected from: (CH2) n , wherein n is an integer ranging from 1 to 10 (including the end value), which is optionally replaced by one or more ZR 10 substituted with a radical, wherein Z is O or S, and R 10 is hydrogen or an alkyl, alkenyl or alkynyl group; or

[0185] {-(CH2) k -Y-(CH2) m} p -, wherein k and m are independently integers ranging from 1 to 10 (inclusive), and p is an integer ranging from 1 to 6 (inclusive), and Y is O, S, CO, COO, CONR 11 NR 11 CO or NR 11 COR 11 N, where R 11 Independent of any other R 11 , is hydrogen or an alkyl group;

[0186] Where R1-R 10 One or more CH2 groups of an alkyl, alkenyl or alkynyl group may be replaced by O, S, SS, CO, COO, NR 12 CO、NR 12 COO or NR 12 CONR 12 Instead, where R 12 Independent of any other R 12 , is hydrogen or an alkyl, alkenyl or alkynyl group; and

[0187] Where R1-R 12 The alkyl, alkenyl or alkynyl groups are optionally substituted with one or more OR 13 、CN、Halogen、N(R 13 )2. Peptide or carbohydrate group substitution, where R 13 Independent of other R 13 , is hydrogen or an alkyl, alkenyl or alkynyl group, and

[0188] wherein at least one of R3 and R4, when present as an alkyl group, is replaced by OR 13 and N(R 13 )2 group substituted.

[0189] The synthesis of these compounds and the method for preparing the lipid compositions incorporating these compounds can be achieved by any method 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 such compounds can be found in, for example, U.S. Patent No. 7,166,745 and PCT Publication WO 00 / 27795, both of which are expressly incorporated by reference in their entirety, as if fully set forth herein.

[0190] In some embodiments of the lipid aggregates of the present invention, particularly preferred but non-limiting ionizable lipids for forming the complex have the structural formula IA:

[0191] and its salt,

[0192] Where R 1 and R 2 R is 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; 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; X a - It is an anion.

[0193] In some embodiments of the lipid aggregates of the present invention, particularly preferred compounds for forming complexes are

[0194] A preferred but non-limiting ionizable lipid has the structural formula IB:

[0195] and its salt,

[0196] Where R 5 and R 8 R is 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; 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; and X b - It is an anion.

[0197] In an embodiment, R 1 and R 2 is independently substituted or unsubstituted alkyl. 1 and R 2 R is independently unsubstituted alkyl. 1 and R 2 are independently unsubstituted C1-C 20 In an embodiment, R 1 and R 2 are independently unsubstituted C5-C 20 In an embodiment, R 1 and R 2 is independently unsubstituted C 10 -C 20 In an embodiment, R 1 and R 2 is independently unsubstituted C 12 -C 18 In an embodiment, R 1 and R 2 is independently unsubstituted C 14 -C 16 In an embodiment, R 1 For unsubstituted C 14 In an embodiment, R 2 For unsubstituted C 14 In an embodiment, R 1 For unsubstituted C 15 In an embodiment, R 2 For unsubstituted C 15 In an embodiment, R 1 For unsubstituted C 16 In an embodiment, R 2 For unsubstituted C 16 In an embodiment, R 1 -(CH2) 13 CH3. In an embodiment, R 2 -(CH2) 13 CH3.

[0198] In an embodiment, R 3 and R 4 is independently hydrogen or substituted or unsubstituted alkyl. 3 and R 4 are independently hydrogen.

[0199] In an embodiment, R 5 , R 6 and R 7are independently hydrogen, substituted or unsubstituted alkyl. 5 , R 6 and R 7 is independently hydrogen or unsubstituted alkyl. 5 , R 6 and R 7 are independently hydrogen or unsubstituted C1-C 20 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C5-C 20 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 10 -C 20 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 12 -C 18 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 14 -C 16 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 14 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 15 In an embodiment, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 16 In an embodiment, R 5 For unsubstituted C 14 In an embodiment, R 7 For unsubstituted C 14 In an embodiment, R 5 -(CH2) 13 CH3. In an embodiment, R 6 is hydrogen. In an embodiment, R 7 -(CH2) 13 CH3.

[0200] In an embodiment, R 8is hydrogen or substituted or unsubstituted alkyl. In an embodiment, R 8 For hydrogen.

[0201] In embodiments, m is an integer from about 1 to 6. In embodiments, m is an integer from about 1 to 5. In embodiments, m is an integer from about 1 to 4. In embodiments, m is an integer from about 1 to 3. In embodiments, m is an integer from 1 to 6. In embodiments, m is an integer from 1 to 5. In embodiments, m is an integer from 1 to 4. In embodiments, m is an integer from 1 to 3. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6.

[0202] In embodiments, n is an integer from about 1 to 6. In embodiments, n is an integer from about 1 to 5. In embodiments, n is an integer from about 1 to 4. In embodiments, n is an integer from about 1 to 3. In embodiments, n is an integer from 1 to 6. In embodiments, n is an integer from 1 to 5. In embodiments, n is an integer from 1 to 4. In embodiments, n is an integer from 1 to 3. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6.

[0203] In one embodiment, R 1 -(CH2) 13 CH3, R 2 -(CH2) 13 CH3, R 3 is hydrogen, R 4 is hydrogen, m is 4 and X a CH3COO - .

[0204] In one embodiment, R 5 -(CH2) 13 CH3, R 6 is hydrogen, R 7 -(CH2) 13 CH3, R 8 is hydrogen, n is 4 and X b - CH3COO - .

[0205] 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:

[0206] Compound 1-1:

[0207]

[0208] Compound 1-2:

[0209]

[0210] Compound 1-3:

[0211]

[0212] Compound 1-4:

[0213]

[0214] Compounds 1-5:

[0215]

[0216] Compounds 1-6:

[0217]

[0218] In some embodiments, the ionizable lipids described in U.S. Pat. No. 9,259,475 and U.S. Pat. No. 10,883,118 are contemplated for use in the compositions and methods of the present invention (where these references are incorporated by reference in their entirety). In some embodiments, such lipids are based on a core of N,N'-disubstituted 2,3-dihydroxy-1,4-butanediamine.

[0219] In some non-limiting embodiments, the lipid composition comprises an ionizable diaminobutane lipid molecule having the structure of Formula II:

[0220] and its salt,

[0221] Each R 1 Independently C1-C 23 Alkyl, C1-C 23 Alkenyl, -(CO)C1-C 23 Alkyl or -(CO)C1-C 23 alkenyl; and each R 2 are independently -CH2-(CHR 3 ) 1-6 -CH2-NHR 4 or -CH2-(CHR 3 ) 0-6 -CH2-OH, where each R 3 are independently H, OH or NH2, R 4 is H or CH3; and R 10 It is H or C1-C8 alkyl.

[0222] 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 It can be -CH2-(CHR 3 ) 1-6 -CH2-NHR 4 And R 3 is H or OH. In some embodiments, at each R 3 No more than 3 R's in the section 3 The group is OH.

[0223] In any of these embodiments, each R 1 can be the same or different, and each R 2 Can be the same or different. In a specific embodiment, one or two R 10 Can be H, or one or two R 10 It can be a C1-C3 alkyl group, for example one or two R 10 It may be methyl.

[0224] In the above specific embodiments, one or two R 1 Can be C 12 -C 20 The alkenyl moiety may be a cis-alkenyl moiety.

[0225] In a specific embodiment, one or both R 2 It can be -CH2-CHOH-(CHR 3 ) 0-5 -CH2-NHR 4 ; For example, one or two R 2 It can be -CH2-CHOH-CH2-NH2. In these compounds, R is advantageously C 14-18 Alkyl or C 14-18 Monounsaturated alkenyl.

[0226] Each R 1 , R 2 and / or each R 10 can be the same or different, so the molecule can be symmetrical or asymmetrical. In a specific embodiment, R 2 It can be C1-C3 alkyl and / or R 1 It is monounsaturated C 12 -C 20 When present, R 1 One or two C 12-C 20 The alkenyl moiety may be cis-alkenyl.

[0227] Also provided are molecules having structural formula IIA:

[0228] And its salt.

[0229] In this structure, each R 1 Independently can be C1-C 23 Alkyl, C1-C 23 Alkenyl, -(CO)C1-C 23 Alkyl or -(CO)C1-C 23 Each R 2 R is independently C1-C6 alkyl or C1-C6 alkenyl, optionally interrupted by up to 2 O atoms. 3 independently may be 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- 6NHC(═NH)NH2,((CH2) m O x ) y (CH2) z O x R 8 or -(CH2) 0-3 Het. Each R 4 independently may be H, C1-C6 alkyl or C1-C6 alkenyl, and each R 5 independently may be 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 23Alkenyl or a peptide containing 1-20 amino acid residues. The peptide advantageously contains a plurality of positively charged amino acid side chains. Thus, for example, the peptide may contain one or more lysine, arginine and / or histidine residues. Other positively charged amino acids may also be used, whether naturally occurring or not. Thus, for example, ornithine, homoarginine and other amino acids containing amines, guanidines; imidazole and other basic heterocycles, etc. may be used. Each m independently may be 2-5, and each n independently may be 2-5, and each x independently may be 0 or 1, each y may be 0-2, and each z may be 1-6. R 6 and R 7 independently may be H, an amine protecting group, -(CO)C1-C 23 Alkyl or -(CO)C1-C 23 Alkenyl, R 8 It may be H, C1-C6 alkyl or C1-C6 alkenyl, and Het may be a 5-7 membered monocyclic basic heterocycle or an 8-11 membered bicyclic basic heterocycle.

[0230] The molecule of formula IIA has a substituent R 1 -R 5 Each or all of R may be independently symmetric or asymmetric; 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.

[0231] In a specific embodiment of Formula IIA, R 2 It can be C1-C3 alkyl and / or R 1 Can be monounsaturated C 12 -C 20 Alkenyl; for example R 1 One or two C 12 -C 20 The alkenyl moiety may be cis-alkenyl.

[0232] In other embodiments of Formula II, R 3 It can be -(CH2) m NR 6 (CH2) n NHR 7 And / or R 5 It can be -(CH2) 2-6 NHR 7 In these or other embodiments, m may be 3, n may be 3, and / or R 5It can be -(CH2)3NHR 7 In these or other embodiments, each R 6 and each R 7 It can be H.

[0233] In other embodiments of Formula II, at least one R 6 or R 7 It can be -(CO)C1-C 23 Alkyl, or -(CO)C1-C 23 alkenyl and the rest are H. In a specific embodiment, each R 1 Can be C 12 -C 20 Alkyl or C 12 -C 20 Monounsaturated olefinic group, each R 2 It can be a C1-C3 alkyl group, each R 3 It 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 olefinic group, each R 2 It can be a C1-C3 alkyl group, each R 3 It can be -(CH2)3NR 6 (CH2)3NHR 7 , each R 4 Can be H; each R 5 It can be -(CH2)3NHR 7 , and at least one R 6 or R 7 It can be -(CO)C1-C 23 Alkyl or -(CO)C1-C 23 and the remainder are H.

[0234] Specific examples of the compound of formula II include compounds having the following structure: and its salt,

[0235] Each R can be, for example but not limited to, C 12-20 Alkyl or C 12-20 and each R' and each R" independently can be but not limited to H, an amine protecting group, -(CO)C1-C 23 Alkyl or -(CO)C1-C23 Alkenyl.

[0236] In these and other embodiments of structure (Formula IIB), R can be C 14 -C 18 Alkyl or C 14 -C 18 and / or each R' and each R" independently may be H, C 14 -C 18 Alkyl or C 14 -C 18 Alkenyl.

[0237] Another specific example of the compound of formula II is a group of compounds having the following structure: and its salt,

[0238] Wherein, for example, R can be but is not limited to C 12-20 and / or each R' and each R" independently can be but not limited to H, an amine protecting group, -(CO)C1-C 23 Alkyl or -(CO)C1-C 23 Alkenyl.

[0239] In these and other embodiments, R can be oleyl and each R′ and each R″ independently can be H or oleoyl. In other embodiments, at least one R′ or R″ can be oleoyl and the rest are H.

[0240] In other specific embodiments of structure (II), each R 3 independently may 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 1-20 amino acid residues.

[0241] In these and other embodiments, each R 1 Can be C 12 -C 20 Alkyl or C 12 -C 20 Alkenyl, each R 2 may be C1-C3 alkyl, and / or each R 5 Can be H. In other embodiments, each R 3 It can be -(CH2) 2- 6NH2, -(CH2)3-6 NHC(═NH)NH2 or each R 3 It can be -(CH2) 1-3 In a specific embodiment, Het can be

[0242]

[0243] In some embodiments of the lipid compositions of the present invention, 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 olefinic group:

[0244] Compound 2-1:

[0245]

[0246] Compound 2-2:

[0247]

[0248] Compound 2-3:

[0249]

[0250] Compound 2-4:

[0251]

[0252] Compound 2-5:

[0253]

[0254] Compound 2-6:

[0255]

[0256] Compound 2-7:

[0257]

[0258] Compound 2-8:

[0259]

[0260] Compound 2-9:

[0261]

[0262] Compound 2-10:

[0263]

[0264] In some embodiments, lipids described in US Pat. No. 8,759,499 (the contents of which are incorporated herein by reference in their entirety) are contemplated for use in the compositions and methods of the present invention.

[0265] In some non-limiting embodiments, the lipid composition can include at least a first ionizable lipid and optionally at least a first neutral lipid, wherein the lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein the ionizable lipid has a structure of Formula III:

[0266]

[0267] (Formula III) and salts thereof.

[0268] In the compound of formula III, X1 and X2 can be independently selected from (CH2) n 、(CHOH) n and CONH. X5 and X6 can independently be (CH2) 1-6 W1 and W2 can be independently selected from 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 R3 and R6 can be independently selected from N, NH, CH, N(CH2) s CH3, (CH) n 、(COH) n , CON- and q = 0-1. R4 and R5 can be independently selected from (CH2) n 、(CH2-CHOH-CH2) n 、(CHOH) n , HNCO, CONH, CO, -O-, -S-, -SS-, polyamide and ester bonds. L1 and L2 can be independently selected from the group consisting of -NH-, -O-, -NHCO-, -CONH-, -OCO-, -COO-, -CO-, -S-, -SS-, -NHC(O)O-, -OC(O)NH-, -NHCONH-, -NHC(═NH)NH-, -S(O)- and -SO2-.

[0269] Y is a heterocyclic moiety containing at least one amine or amide moiety. The attachment point of Y can be carbon and / or heteroatom. Examples of suitable heterocyclic moieties include, but are not limited to, piperazine, piperidine, pyridine, pyrrolidine, and imidazole moieties and their derivatives. In a specific embodiment, the heterocyclic moiety is a piperazine ring, wherein the attachment point is optionally at one or two N atoms. The heterocyclic moiety may be optionally substituted with up to 4 substituents independently selected from the group consisting of OH, ═O, carboxylic acid, ether, polyether, alkylaryl, amino alcohol, amide, linear alkyl, branched alkyl, cycloalkyl, linear alkenyl, branched alkenyl, cycloalkenyl, linear 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, alkylpolyamine, alkenylpolyamine, alkynylpolyamine, spermidine, spermine, carboxyspermine, guanidinium, pyridinium, pyrrolidinium, piperidinium, piperazinium, and aminoacyl, wherein 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,

[0270] R1 and R2 independently can be 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, alkylpolyamine, alkenylpolyamine, alkynylpolyamine, spermidine, spermine, carboxyspermine, guanidinium, pyridinium, pyrrolidinium, piperidinium, piperazinium, aminoacyl, peptidyl and protein. In the context of the present invention, it is to be understood that, unless otherwise specifically stated, alkylamine can be an amine containing a short alkyl chain or a long alkyl chain. Similarly, alkenylamine will be understood to contain a short alkenyl chain or a long alkenyl chain, as is the case with alkynylamine.

[0271] Z1 and Z2 can independently be selected from the group consisting of straight chain alkyl, branched chain alkyl, cycloalkyl, straight chain alkenyl, branched chain alkenyl, cycloalkenyl, straight chain alkynyl, branched chain alkynyl, wherein m, n, p and s are independently 0-6, provided that when m, n and p are all 0, then Y is eliminated and R3 is directly bonded to X2.

[0272] Y can have the following ring structure

[0273]

[0274] Wherein X3 and X4 can be independently selected from N and CH, and n1 and n2 are independently 1-10.Usually, Y is a 6-9 membered ring, and in an exemplary specific embodiment, X3 and X4 are both N and n1 and n2 are both 2, i.e., Y is an optionally substituted piperazine moiety.This structure can be optionally substituted by 1-4 moieties, as described above for Y.

[0275] In other specific embodiments, Y may have the following cyclic structure:

[0276]

[0277] wherein n1 and n2 are independently 1 to 10. Typically, n1+n2 is 3 to 7. Such cyclic structures may be optionally substituted with 1 to 4 independently selected moieties, as described above for Y.

[0278] An example of a lipid can be defined by the following structure, where both L1 and L2 are NH and X5 and X6 are CH2:

[0279] And its salt.

[0280] In this structure, R1-R6, W1, W2, X1, X2, Z1, Z2, Y, m, p and q are as defined above.

[0281] 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, wherein R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CHOH-CH2; R3, R6 = N; Z1, Z2 = oleoyl; W1, W2 = H; q, p, m = 1; and Y = piperazine. Other examples include ((bis(3-{N-3-aminopropyl-N-palmityl}amino-2-hydroxypropyl)-piperazine)) and other compounds where the alkyl group length varies from C12 to C18, including but not limited to those 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 both Z1 and Z2 are palmityl, myristyl, lauryl or stearyl.

[0282] Other specific examples of compounds of formula III for use herein include, but are not limited to, the following:

[0283] Compound 3-1: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CHOH-CH2; R3, R6 = N; Z1, Z2 = palmityl; W1, W2 = H; q, p, m = 1; and Y = piperazine;

[0284] Compound 3-2: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CHOH-CH2; R3, R6 = N; Z1, Z2 = myristyl; W1, W2 = H; q, p, m = 1; and Y = piperazine;

[0285] 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; and Y = piperazine;

[0286] 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; and Y = piperazine;

[0287] 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; and Y = piperazine;

[0288] Compound 3-6: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CHOH-CH2; R3, R6 = N; Z1, Z2 = palmityl; W1, W2 = OH; q, p, m = 1; and Y = piperazine;

[0289] Compound 3-7: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CHOH-CH2; R3, R6 = N; Z1, Z2 = myristyl; W1, W2 = OH; q, p, m = 1; and Y = piperazine;

[0290] 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; and Y = piperazine;

[0291] 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; and Y = piperazine;

[0292] 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; and Y = piperazine;

[0293] Compound 3-11: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CH2-CH2; R3, R6 = N; Z1, Z2 = palmityl; W1, W2 = H; q, p, m = 1; and Y = piperazine;

[0294] Compound 3-12: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CH2-CH2; R3, R6 = N; Z1, Z2 = myristyl; W1, W2 = H; q, p, m = 1; and Y = piperazine;

[0295] 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; and Y = piperazine;

[0296] 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; and Y = piperazine;

[0297] 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; and Y = piperazine:

[0298] Compound 3-16: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CH2-CH2; R3, R6 = N; Z1, Z2 = palmityl; W1, W2 = OH; q, p, m = 1; and Y = piperazine;

[0299] Compound 3-17: R1, R2 = H; X1, X2 = CH2; R4, R5 = CH2-CH2-CH2; R3, R6 = N; Z1, Z2 = myristyl; W1, W2 = OH; q, p, m = 1; and Y = piperazine;

[0300] 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; and Y = piperazine;

[0301] 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; and Y = piperazine.

[0302] In the context of the provided lipids, unless otherwise defined, short chain alkyl groups are typically C1-C6 alkyl groups. Unless otherwise defined, long chain alkyl groups are typically C 10 -C 20 Alkyl or C10 -C 30 Alkyl. When not specifically defined, either definition may be used appropriately. Unless otherwise defined, those skilled in the art will also understand that other derivative groups containing alkyl moieties (e.g., alkoxy moieties, etc.) may also contain short-chain and / or long-chain groups as appropriate in the context. Alkenyl groups contain at least one cis or trans carbon-carbon double bond and are typically C 10 -C 30 Exemplary alkenyl groups contain one or two cis double bonds, wherein the double bonds are disubstituted. Alkynyl groups contain at least one carbon-carbon triple bond and are typically C 10 -C 30 The alkyl, alkenyl or alkynyl groups may be straight chain or branched. Unless otherwise defined, it will be understood by those skilled in the art that other derivative groups containing alkyl moieties (e.g., alkoxy moieties, etc.) may also contain short chain and / or long chain groups as appropriate in the context.

[0303] In some non-limiting embodiments, ionizable lipids contemplated for use in the compositions and methods of the present invention include lipid molecules having the structure of Formula IV:

[0304] And its salt.

[0305] In this structure, L1 and L3 are independently absent or independently can be: -CO q -, C3-C8 alkyl optionally inserted with N, O or -C(O)O-; monounsaturated C4-C8 alkenyl, -CO optionally inserted with N, O or -C(O)O- q C3-C8 alkyl; and -CO q -monounsaturated C4-C8 alkenyl; wherein q is 1 or 2; or when R 1 and R 2 If it is not H, then L1X and L3Y can be -CR independently. 3 =C(R 4 )R 5 . L2 is C4-C 12 Alkylene, optionally substituted at up to 2 positions with OR 9 replace.

[0306] X and Y independently may be selected from:

[0307] L4-Het, wherein L4 is a C1-C4 alkylene group and Het is a C4-C4 alkylene group containing at least one nitrogen atom. 12 Heterocyclic ring;

[0308]

[0309] and

[0310]

[0311] R 1 and R 2 independently can be H, C8-C 20 Alkyl or monounsaturated C8-C 20 Alkenyl. 3 To R 8 can be independently selected from H, -C1-C6 alkyl- and C3-C6 cycloalkyl. In a specific example, R 3 To R 8 are independently H or C1-C3 alkyl.

[0312] R 9 It can be H, -CO-C8-C 20 Alkyl, -CO-monounsaturated C8-C 20 Alkenyl, C8-C 20 Alkyl or monounsaturated C8-C 20 Specific examples include: 9 It is C 14 -C 18 Alkyl or monounsaturated alkenyl; wherein R 9 is H, and R 1 and R 2 Not H; where R 9 Not H, and R 1 and R 2 is H; and where R 9 It is C 14 -C 18 Alkyl or -CO-C 14 -C 18 Alkyl, and R 1 and R 2 is H. Advantageously, when present, R 9 The double bond in is a cis double bond.

[0313] In these molecules, X and Y may be the same or different, L1 and L3 independently may be the same or different, and R 1 and R 2 Can be the same or different.

[0314] In a specific embodiment, L2 can be C4-C 12 In other embodiments, L2 is advantageously -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p-, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8. In a specific example, L2 is -CH2CH(OR 9 )CH(OR 9 )CH2-.

[0315] In other embodiments, the molecule may contain a heterocyclic moiety linked via an amide bond or a carbamate bond, wherein L1 is -CO- or -CO 2- , and X is L4Het, wherein Het is a heterocycle as defined below. In yet another embodiment, when R 1 and / or R 2 Instead of H, the molecule may contain an enamine moiety where L1X is -CR 5 =C(R 3 )R 4 In such enamine moieties, R3 and R5 may together form a C3-C7 carbocyclic ring.

[0316] The molecule of formula IV has a substituent R 1 , R 2 , L 2-3 and each or all of X and Y may independently be symmetric or asymmetric; that is, each R 1 can be the same or different, each R 2 can be the same or different, L1 and L3 can be the same or different R 3 , and X and Y may be the same or different. In addition, the structure of L2 does not have to be symmetrical.

[0317] Specific examples of compounds of formula IV used herein include: Compound 4-1:

[0318]

[0319] Compound 4-2:

[0320]

[0321] Compound 4-3:

[0322]

[0323] Compound 4-4:

[0324]

[0325] Compound 4-5:

[0326]

[0327] Compounds 4-6:

[0328]

[0329] Compounds 4-7:

[0330]

[0331] Compounds 4-8:

[0332]

[0333] Compounds 4-9:

[0334]

[0335] Compounds 4-10:

[0336]

[0337] Compounds 4-11:

[0338]

[0339] Compounds 4-12:

[0340]

[0341] Compounds 4-13:

[0342]

[0343] Compounds 4-14:

[0344]

[0345] Among these molecules, R 1 or R 2 Each for example may be but is not limited to C 14-18 alkyl or C 14-18 alkenyl; and R 9 Each may independently be, but is not limited to, H, -(CO)C 14 -C 18 Alkyl or -(CO)C 14 -C 18 alkenyl.

[0346] In some embodiments, the lipid described in U.S. Patent No. 8,034,977 is contemplated for use in the compositions and methods of the invention (the contents of that patent are incorporated herein by reference in their entirety).

[0347] In some non-limiting embodiments, the lipid composition can comprise at least a first ionizable lipid and optionally at least a first neutral lipid, wherein the lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein the ionizable lipid has a structure of Formula (V):

[0348] or a salt thereof,

[0349] wherein X is N(R4R5R6) or a dialkylphosphatidyl group, or X is

[0350]

[0351] In formula V, Y and Z independently may be selected from the group consisting of alkoxy, alkanoyloxy, alkylamine, alkylcarbamate, and alkylguanidine. 1 and R 2 R may be independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkylamine, alkylamino alcohol, sperminyl, spermidine and carboxysperminyl. 3 is H or C1-C4 alkyl. 4 , R 5 and R 6 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl and alkylaryl, provided that R 4 , R 5 and R 6 At least one of W is a long chain alkyl or alkenyl. Alkyl, alkenyl, aryl and alkylaryl groups may contain, for example, 6 to 30 carbon atoms, advantageously 10 to 18 carbon atoms, although those skilled in the art will recognize that these groups may contain less than 6 or more than 30 carbon atoms. W may be a short chain (C1-C6) alkyl or alkylamino, and R 7 It can be a negatively charged or short chain (C1-C6) alkyl group.

[0352] Examples of compounds of formula V are the group of compounds having formula VA:

[0353] and its salt,

[0354] Where each R is independently C1-C 30 Alkyl, alkenyl or alkanoyl;

[0355] Z3 and Z4 are independently C1-C6 alkyl;

[0356] R 1 and R 2 independently selected from the group consisting of hydrogen, C1-C6 alkyl, alkylamine, alkylamino alcohol, sperminyl, spermidine and carboxysperminyl,

[0357] R3 is H or C1-C4 alkyl, and

[0358] R' is H or

[0359] Specific examples of alkylamino alcohol lipids of Formula VA include, but are not limited to:

[0360] Compound 5-1:

[0361]

[0362] Compound 5-2:

[0363]

[0364] , where each R is independently C 10 -C 18 Alkyl or alkenyl.

[0365] In some embodiments, additional ionizable lipids (Compounds X-1 to X-16) contemplated for use in the compositions and methods of the present invention are provided below, as well as Figures 1 and 2 from Han et al. (2021); "An ionizable lipid toolbox for RNA delivery; Vol. 12, p. 7233. Figure 2 Any other lipid of the invention, which is incorporated by reference in its entirety.

[0366] Compound X-1

[0367]

[0368] Compound X-2

[0369]

[0370] Compound X-3

[0371]

[0372] Compound X-4

[0373]

[0374] Compound X-5

[0375]

[0376] Compound X-6

[0377]

[0378] Compound X-7

[0379]

[0380] Compound X-8

[0381]

[0382] Compound X-9

[0383]

[0384] Compound X-10

[0385]

[0386] Compound X-11

[0387]

[0388] Compound X-12

[0389]

[0390] Compound X-13:

[0391]

[0392] Compound X-14

[0393]

[0394] Compound X-15

[0395]

[0396] Compound X-16

[0397]

[0398] The central amine moiety of the lipids 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. Therefore, the lipid can have a positive charge or a partial positive charge at physiological pH.

[0399] Those skilled in the art will recognize that although some ionizable lipid molecules are shown here in their neutral (unprotonated) form for convenience, these molecules will exist in partially or fully protonated form in solution at an appropriate pH, and the present invention includes all of their protonated, unprotonated, ionized and non-ionized forms without limitation, unless otherwise specifically stated.

[0400] Advantageously, the lipid composition comprises one or more neutral co-lipids in addition to the ionizable lipids, although those skilled in the art will appreciate that other co-lipids may be used. The neutral lipid may, for example, be selected from the group consisting of a sterol or sterol derivative, a phospholipid or a combination thereof.

[0401] The neutral lipid may be present at about 5 mol%-60 mol% of the total lipid preparation. In some embodiments, the neutral lipid is present at about 15 mol%-50 mol%, such as 25 mol%-40 mol%. In certain embodiments, the amount of neutral lipid in the lipid compositions disclosed herein is at least about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or 60 mol% of the total lipid preparation.

[0402] In some embodiments, the lipid composition includes neutral lipids, and the neutral lipids include cholesterol. In some embodiments, the neutral lipids include sterols. In some embodiments, the neutral lipids include dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the neutral lipids include diphytanoylphosphatidylethanolamine (DPhPE). In some embodiments, the neutral lipids include Lyso-PE (1-acyl-2-hydroxyl-sn-glycero-3-phosphoethanolamine). In some embodiments, the neutral lipids include Lyso-PC (1-acyl-3-hydroxyl-sn-glycero-3-phosphocholine). In some embodiments, the neutral lipids include distearoylphosphatidylcholine (DSPC). In some embodiments, the neutral lipids include dioleoylphosphatidylcholine (DOPC). In some embodiments, the neutral lipids include dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the neutral lipids include palmitoyloleoylphosphatidylcholine (POPC). In some embodiments, the neutral lipids include palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). In some embodiments, the neutral lipids include dipalmitoylphosphatidylethanolamine (DPPE). In some embodiments, the neutral lipids include dimyristoylphosphoethanolamine (DMPE). In some embodiments, the neutral lipids include distearoyl-phosphatidylethanolamine (DSPE). In some embodiments, the neutral lipids include 16-O-monomethyl PE. In some embodiments, the neutral lipids include 16-O-dimethyl PE. In some embodiments, the neutral lipids include 18-1-trans PE. In some embodiments, the neutral lipids include 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE). In some embodiments, the neutral lipids include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In some embodiments, the neutral lipids include any combination thereof.

[0403] Exemplary phospholipids that can be used in the compositions disclosed herein include, but are not limited to, 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 (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-phosphoethanolamine (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 hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), or any combination thereof.

[0404] The phospholipid that can be used in the compositions provided herein can for example exist with about 5 mol % to about 20 mol % of lipid composition preparation. Advantageously, phospholipid exists with about 1 mol % to about 40 mol %, for example, 1 mol % to about 25 mol % scope. Preferably, the amount of phospholipid in lipid composition preparation 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 total lipid composition preparation, or any amount therebetween.

[0405] In some embodiments, the phospholipids that can be used in the composition can be present at about 5 mol% to about 15 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 5 mol% to about 10 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 5 mol% to about 12 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 35 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 30 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 25 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 20 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 15 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 10 mol%. In some embodiments, the phospholipids that can be used in the composition can be present at about 1 mol% to about 5 mol%.

[0406] In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 0.5 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 1 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 2 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 3 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 4 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 5 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 10 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 12 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 15 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 16 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 17 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 18 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 19 mol%. In some embodiments, the amount of phospholipids in the lipid composition formulations disclosed herein is at least about 20 mol%.

[0407] Other neutral lipids that can be advantageously included in the lipid composition preparations provided herein include sterols or lipids containing sterol moieties ("sterol derivatives"). As defined herein, "sterols" are the steroid subgroups consisting of steroid alcohols. Exemplary sterols that can be used in the lipid composition preparations provided herein and lipids containing sterol moieties include but are not limited to cholesterol, coprosterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterols, tomatine, tomato, ursolic acid, α-tocopherol, hopanes, phytosterols, steroids and mixtures thereof. In some embodiments, structural lipids are sterols. Some lipid composition preparations provided herein include sterols or sterol derivatives. Sterols or sterol derivatives can exist with about 5 mol%-60 mol% of the total lipid composition preparation. Advantageously, sterols or sterol derivatives exist with about 15 mol%-50 mol%, for example, 25 mol%-40 mol%. Preferably, the amount of sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol% or 60 mol% of the total lipid preparation. Some lipid composition formulations provided herein do not comprise a sterol or a sterol derivative.

[0408] The lipid complex compositions (e.g., lipid nanoparticles, liposomes, and liposome complexes) provided herein may also include one or more compounds and / or compositions comprising cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine, polyornithine, and polyarginine, polyamidoamines, poly(β-amino esters), oligoalkylamines, positively charged dendrimers and broken dendrimers, cationic β-cyclodextrin-containing polymers (CD-polymers), DEAE-dextran, etc. The lipid complex compositions may include linear PEI, branched PEI, and / or their derivatives or modified forms, such as cyclodextrin-PEI, stearic acid-PEI, aromatic-PEI, histidyl-PEI, and PEI-PEG. Linear PEI, branched PEI, and their derivatives of various molecular weights are commercially available.

[0409] The lipoplex compositions provided herein (eg, lipid nanoparticles, liposomes, and lipoplexes) can also be combined with one or more exosomes or biological materials (eg, lipids, proteins, nucleic acids, etc.) derived from or purified from exosomes.

[0410] The term "ectosome" refers to a small membrane vesicle secreted by a large portion of a cell that contains a cell-specific payload of proteins, lipids, and genetic materials and other biomolecules that are transported to other cells at different locations in the tissue. The ectosome can be considered a liposomal particle. The ectosome or lipid mixture obtained therefrom can be used in combination with other transfection agents or auxiliary lipid mixtures. The ectosome is also known as a microvesicle, epididymal body, acrosome, ectosome-like vesicle, microparticle, prosome, prostate body, exosome, tumor cell extracellular body, archaeal cell body and cancer body.

[0411] Examples of lipid components isolated from exosomes include, but are not limited to, Lyso-PC (non-limiting examples of which are C-18, C-16, C-14 and mixtures), Lyso-bisphosphatidic acid (non-limiting examples of which are C-18, C-16 and C-14), sphingomyelin, ceramide (non-limiting examples of which are C-8-C-24), disaturated PC (non-limiting examples of which are DSPC, DPPC, DMPC and other examples where Cn (n=8-25)), di-unsaturated PC-MIX (non-limiting examples of which are DOPC, DP(db)PC), phosphatidylserine (PS), phosphatidylinositol (PI), disaturated PE (non-limiting examples of which are DSPE, DPPE, DMPE), di-unsaturated PE-MIX (non-limiting examples of which are DOPE, DP(db)PE), phosphatidylglycerol (PG) (non-limiting examples of which are C-18-C-22), cholesterol and diacylglycerol (such as cardiolipin).

[0412] Also contemplated are any mixtures of combinations of the above listed ionizable lipids, neutral lipids, cationic polymers, exosomes, and / or lipid mixtures isolated from exosomes.

[0413] The lipid compositions provided herein may also include stabilizers, such as stable lipids.Stable lipids may be neutral lipids, or they may be charged.Stable lipids that may be advantageously used in the preparations provided herein include, but are not limited to, lipids modified by polyethylene glycol (PEG).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-diacyloxypropane-3-amine. Such lipids are also referred to as PEGylated lipids.For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.Other stable lipids that may be used in the compositions disclosed herein include, for example, polyethylene glycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers and amphiphilic branched polymers. In an embodiment, the stabilizer can 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 octadecene-9-enoate), Myrj52 (polyoxyethylene (40) stearate), Brij TM S10 (polyoxyethylene (10) stearyl ether), BRIJ TM L4 = polyoxyethylene (4) lauryl ether; BRIJ TM S20 = polyoxyethylene (20) stearyl ether; BRIJ TMS35 = polyoxyethylene (23) lauryl ether; TPGS1000 = D-α-tocopheryl polyethylene glycol 1000 succinate; Tween 20 / polysorbate 80 / tridecyl-D-maltoside in equal proportions and combinations thereof. In certain compositions, the stabilizer is present at about 0.1 mol%-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.

[0414] In some embodiments of lipid compositions containing a payload, the payload is compounded to the outside of a lipid complex (e.g., a liposome, a lipid nanoparticle). In some embodiments of lipid compositions containing nucleic acids, the nucleic acids are compounded to the outside of a lipid complex (e.g., a liposome, a lipid nanoparticle). In some embodiments, the composition has about 20% to about 50% of nucleic acids compounded to the outside of 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 nucleic acids compounded to the outside of the lipid complex. The external compounding of nucleic acids can be measured by methods known in the art, such as Blakney et al. (2019) Gene Therapy 26:363-372.

[0415] In some embodiments of lipid compositions containing a payload, the payload is compounded inside a lipid complex (e.g., liposomes, lipid nanoparticles). In some embodiments of lipid compositions containing nucleic acids, the nucleic acids are compounded inside a lipid complex (e.g., liposomes, lipid nanoparticles). 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%.

[0416] Encapsulation efficiency (EE%) can be measured using a fluorescence plate-based assay using RiboGreen reagent. The assay measures the amount of mRNA in samples with intact LNPs to determine the amount of unencapsulated RNA, and measures the amount of mRNA in LNP samples damaged by triton X-100 to measure total RNA. Encapsulation efficiency % is calculated as the difference between total RNA and unencapsulated RNA divided by total RNA.

[0417] The encapsulation efficiency of the therapeutic and / or prophylactic agent describes the amount of the therapeutic and / or prophylactic agent encapsulated or otherwise associated with the lipid composition after preparation relative to the initial amount provided. In some embodiments, the encapsulation efficiency is expected to be high (e.g., close to 100%). For example, before and after the lipid complex composition is decomposed with one or more organic solvents or detergents, the encapsulation efficiency can be measured by comparing the amount of the therapeutic and / or prophylactic agent in the solution containing the lipid complex composition. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agents (e.g., RNA) in the 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%, such as 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 may be at least 90%.

[0418] Methods of inducing an immune response

[0419] Provided herein are methods for inducing an immune response in a subject, the method comprising administering to the subject a lipid composition as described herein. In some embodiments, the lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, and a nucleic acid molecule comprising a charge (P), wherein the peptide comprises LLELLESL (SEQ ID NO: 6), wherein the composition has 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, and a nucleic acid molecule comprising a charge (P), wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), wherein the composition has 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 has 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 has 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 lipid composition administered further comprises at least one neutral lipid. In some embodiments, the administered lipid composition further comprises a transfection enhancing agent, such as a peptide comprising SEQ ID NO:25 and / or SEQ ID NO:26.

[0420] In some examples, administration of the composition includes systemic administration or topical administration. For example, topical administration includes intramuscular administration or subcutaneous administration. In some embodiments, administration includes administration to the brain, spinal cord, eye (e.g., intravitreal administration) or lymph node (e.g., intranodal administration) of the subject. In other examples, administration may include ex vivo administration, wherein immune cells (e.g., dendritic cells) are ex vivo targeted. In an example, the subject is a mammalian subject.

[0421] Also provided herein is a method for delivering a lipid composition to an immune cell of a subject, wherein the method comprises administering a lipid composition as described herein. In some embodiments, the lipid composition comprises at least one ionizable lipid having a charge (N), at least one peptide, and a nucleic acid molecule comprising a charge (P), wherein the peptide comprises LLELLESL (SEQ ID NO: 6), wherein the composition has 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 administered comprises at least one ionizable lipid having a charge (N), at least one peptide, and a nucleic acid molecule comprising a charge (P), wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), wherein the composition has 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 has 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 has 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 lipid composition administered further comprises at least one neutral lipid. In some embodiments, the administered lipid composition further comprises a transfection enhancing agent, such as a peptide comprising SEQ ID NO:25 and / or SEQ ID NO:26.

[0422] In addition, provided herein is a method for targeting a payload to an immune cell of a subject, the method comprising administering a lipid composition containing a payload as described herein. For example, immune cells include T cells, B cells, dendritic cells (DC), T helper cells, cytotoxic T cells (CTL), natural killer cells (NK), macrophages (including tissue-specific macrophage colonies) or combinations thereof. In some examples, immune cells include DC. In other examples, immune cells include spleen immune cells.

[0423] In some embodiments, the lipid composition targets the immune cells of the subject of the subject by 1.2 times or more compared to the non-immune cells of the subject being targeted. In some embodiments, the lipid composition targets the immune cells of the subject of the subject by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times or more compared to the non-immune cells of the subject being targeted.

[0424] Relative to control level, determined level can be the level of increase.As used herein, the term "increase" relative to level (for example, protein or mRNA level) refers to any % increase higher than control level.In various embodiments, relative to control level, the level of increase can be at least or about 5% increase, at least or about 10% increase, at least or about 15% increase, at least or about 20% increase, at least or about 25% increase, at least or about 30% increase, at least or about 35% increase, at least or about 40% increase, at least or about 45% increase, at least or about 50% increase, at least or about 55% increase, at least or about 60% increase, at least or about 65% increase, at least or about 70% increase, at least or about 75% increase, at least or about 80% increase, at least or about 85% increase, at least or about 90% increase, at least or about 95% increase.

[0425] Relative to control level, determined level can be the level of reduction.As used herein, the term " reduction " relative to level (for example, protein or mRNA level) refers to any % reduction below control level.In various embodiments, relative to control level, the level of reduction can be at least or about 5% reduction, at least or about 10% reduction, at least or about 15% reduction, at least or about 20% reduction, at least or about 25% reduction, at least or about 30% reduction, at least or about 35% reduction, at least or about 40% reduction, at least or about 45% reduction, at least or about 50% reduction, at least or about 55% reduction, at least or about 60% reduction, at least or about 65% reduction, at least or about 70% reduction, at least or about 75% reduction, at least or about 80% reduction, at least or about 85% reduction, at least or about 90% reduction, at least or about 95% reduction.

[0426] For example, in biodistribution studies, female BALB / c mice of 6 weeks of age-10 weeks of age receive lipid formulations containing 5 μg firefly luciferase mRNA via intramuscular (IM) or intravenous (IV) approaches, respectively. 4 hours after injection, mice were injected with d-luciferin (150 mg kg-1, intraperitoneal) and imaged using IVIS Lumina III system (Perkin Elmer) to observe the signal of luciferase. In order to further observe the signal value of a specific organ, tissues including liver, spleen, muscle and lymph node were collected immediately, and the luminescence of tissue samples was measured by IVIS Lumina III system. The luminous intensity in each region of interest (ROI) of the separated organ was quantitatively measured using Living Image 3.0 software (PerkinElmer, Waltham, USA).

[0427] Additionally, targeted delivery of immune cells to the spleen included biodistribution (as described above), which demonstrated efficient delivery specifically to the spleen following intravenous administration of luciferase mRNA. Targeted delivery was also assessed using reporter mRNA and efficiency analysis by flow cytometry.

[0428] In some embodiments, a method for delivering a payload to a subject's spleen cells 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 a payload, to form a lipid complex; and (ii) administering the lipid complex containing the payload to the subject. In some embodiments, a method for delivering a nucleic acid to a subject's spleen cells is provided, the method comprising: (i) mixing at least one ionizable lipid, at least one peptide, wherein the peptide has at least 80% sequence identity with GLFEALLELLESLWELLLEA (SEQ ID NO: 6); and a 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.

[0429] In other examples, methods are provided for expressing a protein in spleen tissue of a subject, the method comprising administering a lipid composition (lipoplex) to the subject.

[0430] Preparation method

[0431] In some embodiments, a method for preparing a population of lipid preparations containing nucleic acid payload molecules is provided, the method comprising (a) mixing a nucleic acid payload with a peptide in an aqueous solution, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1); (b) injecting a lipid solution comprising an ionizable lipid into the aqueous solution, wherein the injection comprises extrusion, online mixing, microfluidic mixing, evaporation or vortexing; and (c) producing a population of lipid preparations complexed with the nucleic acid payload. In some embodiments, when added, the peptide is in an ethanol solution. In some embodiments, the lipid solution of (b) further comprises at least one neutral lipid. In some embodiments, the peptide has 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 NO: 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.

[0432] 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, wherein the lipid phase comprises an ionizable lipid, (b) contacting the components of step (a) with the 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) producing a population of lipid formulations complexed with the nucleic acid payload. In some embodiments, when mixed with an aqueous solution containing nucleic acid molecules, the peptide and lipid are in an ethanol solution. In some embodiments, the lipid phase of (a) further comprises at least one neutral lipid. In some embodiments, the peptide has 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 NO: 6-24. In some embodiments, step (a) further comprises contacting the component with a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO: 26.

[0433] In some embodiments, the preparation method produces a population of lipid nanoparticles in which a payload molecule is encapsulated. In other embodiments, the preparation method produces a population of liposomes in which a payload is encapsulated. In some embodiments, the preparation method produces a population of lipid nanoparticles with a payload molecule compounded to the outside of the lipid nanoparticle. In other embodiments, the preparation method produces a population of liposomes with a payload molecule compounded to the outside of the liposome. In some embodiments, the payload is a nucleic acid, such as an RNA or DNA molecule. In other embodiments, the payload includes a peptide or polypeptide molecule.

[0434] Reagent test kit

[0435] In various aspects, a kit comprising a lipid composition is provided. In embodiments, the kit comprises at least one ionizable lipid and at least one peptide and an agent, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1). In some embodiments of the kit, the peptide has at least 80% sequence identity to 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 (e.g., local injection) to a subject.

[0436] The present invention also provides packaging and kits containing the pharmaceutical compositions for use in the methods of the present invention. The kit may include one or more containers selected from the group consisting of bottles, vials, ampoules, blister packs and syringes. The kit may also include one or more instructions for treating and / or preventing the diseases, conditions or disorders of the present invention, one or more syringes, one or more applicators or a sterile solution suitable for reconstitution of the pharmaceutical compositions of the present invention.

[0437] In some instances, the kit comprising the lipid composition provides that the ionizable lipid and the neutral lipid are in a separate container from the peptide. In some instances, the kit comprising the lipid composition provides that the ionizable lipid and the neutral lipid are in the same container as the peptide.

[0438] Example

[0439] The following examples illustrate some specific embodiments of the present invention, but are not intended to limit the scope of the present invention. Embodiments herein are further described by the following examples and detailed schemes. However, examples are only intended to illustrate embodiments and should not be construed as limiting the scope of this paper. The contents of all references, published patents and patent applications cited throughout this application are incorporated herein by reference.

[0440] Example 1

[0441] Screening has at least one ionizable lipid and at least one peptide lipid-peptide complex preparation, and uses the RNA payload of complex to be evaluated by various in vivo functional tests.Performance and transfection efficiency analysis include payload delivery, biodistribution, cellular uptake and response to the expression of payload encoded protein.The mRNA used includes those encoding firefly luciferase (fLuc), Cre recombinase, GFP and influenza virus hemagglutinin (HA).

[0442] At least one ionizable lipid, a neutral lipid, and a peptide of SEQ ID NO: 6 are complexed with the mRNA payload using standard complexation or lipid nanoparticle formation procedures. As exemplified herein, the formulations examined vary, for example, in peptide concentration, N / P ratio, and / or the type of ionizable lipid.

[0443] Table 3. Exemplary lipid complex formulations :

[0444] preparation Ionizable lipid types Peptide concentration LP1 IB 0.05mg / ml LP2 Formula II 0.05mg / ml LP3 Formula IA 0.05mg / ml LP4 Formula II (Peptide-free) LP5 Formula II 0.05mg / ml LP6 Formula II 0.1mg / ml LP7 Formula VA (Peptide-free) LP8 Formula VA 0.05mg / ml LP9 Formula VA 0.1mg / ml LP10 Formula VA 0.15mg / ml

[0445] Table 4. Exemplary lipid complex formulations :

[0446] preparation Ionizable lipid types Lipid concentration Peptide concentration LP11 Type IA + Type IB 0.8mg / ml 0.05mg / ml LP12 Formula IA 2mg / ml 0.05mg / ml LP13 Formula IA 2mg / ml 0.1mg / ml LP14 Formula IA 2mg / ml 0.2mg / ml LP15 Formula IA 2mg / ml 0.4mg / ml LP16 Formula II 2mg / ml 0.1mg / ml LP17 Formula II 2mg / ml 0.2mg / ml LP18 Formula II 2mg / ml 0.4mg / ml

[0447] Table 5. Exemplary lipid complex formulations :

[0448]

[0449] The ionizable lipid concentrations in the formulations of Table 3 ranged from 0.8 mg / l to 2.0 mg / l, and all formulations in Tables 3-5 included one or two neutral lipids. The ionizable lipid types refer to the structural formulas provided herein.

[0450] The formulations in the examples were prepared using reverse evaporation or microfluidization. The lipids were weighed and dissolved in chloroform and then evaporated on a rotary evaporator. The lipid film was hydrated with water. In some cases, the liposome formulation was passed through a microfluidizer. The lipid formulation was incubated with the peptide solution overnight and stored at 4°C until use.

[0451] For complexing with nucleic acids, lipid formulations and nucleic acids are diluted in buffer, mechanically mixed by pipetting and / or vortexing, and incubated at room temperature for 10-20 minutes prior to delivery to rodent models or cells.

[0452] Female BALB / c mice aged 6 to 10 weeks were purchased from The Jackson Laboratory and acclimated for 7 days before the study.

[0453] Example 2

[0454] Firefly luciferase mRNA was complexed with each lipid-peptide formulation. Mice were injected with 5 μg of fLuc mRNA formulated lipid complexes using an intravenous tail vein injection in a total volume of 100 ml. Four hours after injection, mice were anesthetized with isoflurane anesthesia and imaged for 10 min after an intraperitoneal injection of 100 μL Rediject D-luciferin (Perkin Elmer). Quantitative bioluminescence imaging was performed in vivo and in vitro using the IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0455] like Figure 1A-Figure 1C As shown, intravenous administration of various lipid complex-peptide formulations resulted in luciferase expression in the spleen. Formulations LP4, LP5, and LP6 all contained the same ionizable lipids and differed only in the amount of peptide. The presence of peptide in the formulation significantly increased the expression of the payload mRNA in spleen tissue compared to lipid formulations without peptide. For example, Figure 1B The flux of LP4 (without peptide) was compared with that of LP5 and LP6.

[0456] Formulations LP7, LP8, LP9 and LP10 contained the same ionizable lipids and differed only in the amount of peptide. Figure 1A and Figure 1B Compared to the formulations shown in Figure 1C The formulations shown in had less overall expression in the spleen, but for the lipid formulations based on Formula VA, the presence of the peptide significantly increased the expression of luciferase mRNA in spleen tissue. Figure 1C The flux of LP7 (without peptide) was compared with that of the formulation.

[0457] The in vivo delivery performance of formulations LP11-LP22 with N / P ratios ranging from 0.1 to 5 was evaluated after intravenous administration. Figure 2 The results of the preparations were compared with the mRNA compound preparation LP11 ( Figure 2 The results were compared with those obtained with "LP11-no peptide" in the previous study.

[0458] The in vivo delivery performance was evaluated after intramuscular administration of formulations LP11-LP22 with N / P ratios ranging from 0.1 to 5. The luciferase signal was localized to the injection area (muscle) and the luciferase flux quantification results were Figure 3 As described above, the preparation LP11 ( Figure 3"LP11-no peptide" in the ) was used as a control. No significant fLuc expression was observed in other organs up to 72 hours after intramuscular injection of the formulation, confirming the highly specific non-liver targeting of the formulation.

[0459] The N / P ratio was varied in fLuc mRNA complexed LP11 and LP12 formulations and the effect on splenic delivery and biodistribution after intravenous injection was evaluated. Figure 4A As shown, these formulations with N / P ratios as low as 0.125 can effectively deliver fLuc mRNA expression in the spleen. These formulations also resulted in delivery specifically to the spleen, rather than the liver or lungs ( Figure 4B ).

[0460] Example 3

[0461] tdTomato reporter mice were used to analyze splenocyte populations targeted by lipid-peptide formulations. A schematic diagram of the workflow and analysis is provided in Figure 5 Cre mRNA was complexed with LP11 and LP12 preparations and the complexes were administered intravenously to Ai14 mice. Five days after injection, the animals were sacrificed and the spleens were harvested.

[0462] For splenocyte analysis, single cells were prepared by passing spleen through a 70 μm cell strainer followed by lysis of blood cells with ACK lysis buffer and washing twice with PBS. Suspension Splenocytes were divided into two staining groups and stained with fluorescent antibodies for CD8 (APC), CD4 (PE) and B220 (FITC) or F480 (APC), CD11b (FITC) and CD11c (PE). The fluorescence of BDK-CART transfection in the subpopulations was detected on LSR-II.UV (BD Biosciences) using the Pacific Blue channel.

[0463] like Figure 6 As shown, the LP11 formulation containing Cre mRNA 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.

[0464] The performance of the LP11 and LP12 formulations in splenocyte delivery was compared to the LP11 and LP12 formulations without the SEQ ID NO: 6 peptide ( Fig. 7A and Figure 7BThe performance of "LP11-peptide-free" and "LP12-peptide-free" in the 100 μg / ml PCR product was compared. The lipid formulation was compounded with a lipid composition formulated with Cre mRNA and administered intravenously to Ai14 mice. Five days after administration, splenocytes were prepared as above and labeled with fluorescent antibodies against various immune cell types. A flow cytometry gating strategy was used to identify cell markers and tdTomato expression.

[0465] Compared to LP11 and LP12 formulations without the SEQ ID NO: 6 peptide, both LP11 and LP12 formulations were more effective in delivering CremRNA to all immune cell types of the spleen analyzed (as indicated by tdTomato expression). Fig. 7A ). Fig. 7A As shown, both LP11 and LP12 formulations were significantly more effective in delivering mRNA to dendritic cells compared to other immune cells of the spleen. Figure 7B Representative flow cytometry gating results of dendritic cell populations (MHCII / CD11c) relative to tdTomato expression are shown.

[0466] Firefly luciferase (fLuc) mRNA was formulated with LP11 preparations at different N / P ratios. Similarly, fLuc mRNA was formulated with LP11 preparations without SEQ ID NO: 6 peptide at the same N / P ratio. As described in Example 2, the composition containing mRNA was administered via intravenous (IV) injection and imaged 4h after injection. Fig. 8A and Figure 8B Exemplary results are shown for LP11 formulations with N / P ratios ranging from 0.25 to 4.0 and LP11 without peptide formulation. At N / P ratios ranging from 1.0 to 4.0, LP11 formulations showed enhancement over LP11 without peptide formulation.

[0467] In a repeated dosing experiment, two doses of the fLuc mRNA composition formulated with LP11 formulation at N / P 2 were administered to 4 mice via IV injection, with a two-week period between the two doses. Mice were imaged 4 h after injection as described in Example 2. Imaging of bioluminescence intensity showed that the administered formulation delivered the fLuc mRNA payload specifically to the spleen. Bioluminescence imaging in the region of interest was used to calculate the total flux (photons / second, p / s) from the expressed fLuc. The average total flux measured after the first injection was 1.103×10 9 p / s, and the average total flux measured after the second injection was 1.072×10 9 p / s. Control mice that received a single dose showed 1.103×10 9The total flux of p / s. Thus, the formulation allows for repeated administration and prime-boost dosing.

[0468] Example 4

[0469] use Fig. 9 and Fig.11 The workflow depicted in was used to examine the intracellular endosomal uptake and release of lipoplexes containing nucleic acids. For this analysis, HEK293 cells were transfected with Cy5-labeled mRNA complexed LP preparations or LP11 without SEQ ID NO:6 peptide preparations and incubated at 37°C and 5% CO2. After 15 minutes, 30 minutes, 60 minutes, and 90 minutes, cells were fixed with 4% formaldehyde and counterstained with DAPI to identify cell nuclei. Confocal laser scanning microscopy (Zeiss) was used for fluorescence imaging.

[0470] Example results of mRNA-lipid complex uptake are shown in Fig. 10A As shown in Figure 2, red fluorescence indicates Cy5-mRNA and blue fluorescence indicates DAPI. Quantification of cellular uptake over time was performed in Fig. 10B As shown in , the LP11 preparation was internalized at an earlier time relative to the LP11 preparation without peptide, indicating a more efficient cellular uptake for the intact LP11 preparation.

[0471] To examine the endosomal escape of the complex, HEK293 cells were transfected with Cy5-labeled mRNA complexed LP preparations or LP11 without SEQ ID NO:6 peptide preparations and incubated for 3 hours at 37°C and 5% CO2. 50 nM LysoTracker TM Green DND-26 (Invitrogen) was added to cells for 30 min and then fixed to identify late endosomes involved in lysosomal degradation ( Fig.11 ). The cells were then fixed and counterstained with DAPI as described above, and images were acquired using a confocal laser scanning microscope.

[0472] Exemplary results of endosomal escape of mRNA-lipid complexes are shown in Fig.12 As shown in the figure, red fluorescence indicates Cy5-mRNA (left two images), green fluorescence indicates late endosomal markers (middle two images), and blue fluorescence indicates DAPI (right two images). LP11 preparations showed more efficient endosomal escape than LP11 preparations without peptide. As shown in the merged image ( Fig.12 , right two panels), there is much less mRNA without LysoTracker staining using the intact LP11 preparation compared to the LP11 preparation without peptide.

[0473] Example 5

[0474] Immunogenicity is defined as the ability of a substance (e.g., a vaccine) to elicit an adaptive immune response. Humoral immunity and cell-mediated immunity are two types of adaptive immune responses that enable the body to defend against foreign substances (antigens) in a targeted manner. 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 detecting a specific antigen. Unlike humoral immunity, cellular immunity does not rely on antibodies. Cell-mediated immunity is mainly driven by the release of mature T cells and various cytokines (e.g., IFN-γ) in response to antigens.

[0475] The humoral and cellular immunogenicity of antigens administered via antigen-encoding mRNA formulated with lipid compositions was evaluated to demonstrate the potential of such formulations for vaccine applications. The workflow and tested compositions for immunogenicity studies are described in Fig.13 Shown in.

[0476] Lipid-mRNA complexes of LP11 or LP12 preparations complexed with H10N8 influenza hemagglutinin (HA) mRNA were prepared. The complexes containing mRNA were administered intramuscularly (IM) or intravenously (IV) to BALB / c mice at 4-week intervals according to a prime-boost immunization schedule. Fig.13 The immunization schedule, mRNA dose and route of administration of the complex, and subsequent assays performed to assess humoral and cellular immune responses are indicated. Fig.13 The relationship between the mRNA doses shown in and the doses shown in Figures 14-15 is as follows: 0.02μg is 0.01mg / kg (mRNA amount / mouse body weight); 1μg is 0.05mg / kg, 5μg is 0.25mg / kg, 10μg is 0.5mg / kg, and 20μg is 1mg / kg.

[0477] Serum IgG antibodies binding to the recombinant HA protein were detected in the mouse sera by enzyme-linked immunosorbent assay (ELISA) at week 3 after the prime injection and week 2 after the boost injection (week 6 in total). The HA-specific IgG response after administration of the composition was Fig.14A (for IM administration) and Fig. 14B (for IV administration). For each composition administered, HA-specific IgG results from week 3 and week 6 serum samples from each mouse are shown.

[0478] To evaluate interferon-γ (IFN-γ)-induced HA antigen-specific cellular responses, splenocytes were isolated from the spleen at the end of the prime-boost immunization regimen (week 6). HA-specific peptide stimulation was used for T cell activation in vitro culture, and the released IFN-γ was detected by enzyme-linked immunospot (ELISPOT) assay. Exemplary results are shown in Figure 15A-Figure 15C Shown in.

[0479] Delivery of HA mRNA using both LP11 and LP12 formulations resulted in antigen-specific humoral and cellular immune responses at all exemplified doses, even at the lowest dose tested.

[0480] Although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0481] Patents and scientific literature referenced herein have established knowledge available to those skilled in the art. All references cited herein, such as U.S. Patents, U.S. Patent Application Publications, PCT Patent Applications designating the U.S., published foreign patents and patent applications are incorporated herein by reference in their entirety. GenBank and NCBI cited herein with accession numbers are submitted to be incorporated herein by reference. All other references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the event of a conflict, this specification sheet (including definitions) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive.

[0482] While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A method for delivering a payload to a spleen cell of a subject, the method comprising: A lipid complex is provided, the lipid complex comprising: at least one ionizable lipid, at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 1), and at least one payload molecule; as well as The lipid complex is administered to a subject.

2. The method of claim 1, wherein the lipid complex further comprises at least one neutral lipid.

3. The method of claim 1 or claim 2, wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

4. The method of claim 1 or claim 2, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24.

5. The method according to any one of claims 1 to 4, wherein the lipid complex further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO:

26.

6. The method of any one of claims 1 to 5, wherein the concentration of the peptide is from about 0.001 mg / mL to about 0.5 mg / mL, or from about 0.05 mg / mL to about 0.5 mg / mL.

7. The method according to any one of claims 1 to 6, wherein the payload molecule is a nucleic acid.

8. The method of claim 7, wherein the at least one ionizable lipid comprises a charge (N), the nucleic acid comprises a charge (P), and the lipoplex has 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.

9. The method of any one of claims 1 to 8, wherein the payload comprises an RNA molecule.

10. The method of claim 9, wherein the RNA molecules comprise more than one mRNA molecule.

11. The method of claim 9, wherein the RNA molecule comprises mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or a combination thereof.

12. The method of claim 11, wherein the RNA molecule comprises a sgRNA molecule.

13. The method of claim 11, wherein the RNA molecule comprises an sgRNA molecule and an mRNA molecule.

14. The method of any one of claims 1 to 13, wherein the payload further comprises a protein.

15. The method of claim 7, wherein the nucleic acid encodes an immunogen.

16. The method of claim 7, wherein the nucleic acid encodes hemagglutinin (HA) or ovalbumin.

17. The method of any one of claims 1 to 16, wherein the ionizable lipid comprises a lipid according to any one of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V), or a combination thereof.

18. The method of any one of claims 1 to 17, wherein the ionizable lipid comprises a lipid according to formula (IA) or formula (IB) or a combination thereof.

19. The method of any one of claims 1 to 17, wherein the ionizable lipid comprises a lipid according to Formula (IIA), Formula (IIB), or Formula (IIC), or a combination thereof.

20. The method according to any one of claims 2 to 19, wherein the 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 (POPC), palmitoyl Oleoyl-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 (trans-DOPE) or a combination thereof.

21. The method of any one of claims 1 to 20, wherein the lipid complex comprises a population of lipid nanoparticles.

22. The method of claim 21, wherein the lipid nanoparticles have a diameter of about 20 nm to about 1 μm.

23. The method of any one of claims 1 to 20, wherein the lipid complex comprises a population of liposomes.

24. The method of any one of claims 1 to 23, wherein the administration comprises systemic administration or local administration.

25. The method of any one of claims 1 to 24, wherein the administering comprises administering to the brain, spinal cord, eye, or lymph node of the subject.

26. The method of any one of claims 1 to 24, wherein the administration comprises intravenous administration, intramuscular administration, or subcutaneous administration.

27. The method of any one of claims 1 to 26, wherein the subject comprises a mammalian subject.

28. The method of any one of claims 1 to 27, wherein the payload is delivered to dendritic cells.

29. The method of any one of claims 1 to 28, wherein the lipoplex targets the subject's spleen cells 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 administering a lipoplex comprising the ionizable lipid and the payload molecule without the peptide.

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

31. The composition of claim 30, wherein the peptide concentration is about 0.05 mg / mL.

32. The composition of claim 30, wherein the payload molecule is a nucleic acid.

33. The composition of claim 32, wherein the at least one ionizable lipid comprises a charge (N), the nucleic acid comprises a charge (P), and the lipoplex has 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.

34. A composition comprising: at least one ionizable lipid, said ionizable lipid comprising a charge (N), at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 1), and at least one payload comprising a nucleic acid, wherein the nucleic acid comprises a charge (P), wherein the composition has 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.

35. A composition comprising: at least one ionizable lipid, said ionizable lipid comprising a charge (N), at least one endosomally released peptide, and at least one payload, the payload comprising a nucleic acid, the nucleic acid comprising a charge (P), wherein the composition has an N / P ratio of about 0.01 to about 0.

5.

36. A composition according to any one of claims 30 to 35, further comprising at least one neutral lipid.

37. The composition of any one of claims 30 to 36, wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

38. The composition of any one of claims 30 to 36, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24.

39. The composition of any one of claims 30 to 38, wherein the lipid complex further comprises a second peptide comprising at least one of SEQ ID NO: 25 and / or SEQ ID NO:

26.

40. The composition of any one of claims 34 to 39, wherein the concentration of the peptide is from about 0.001 mg / mL to about 0.5 mg / mL, or from about 0.05 mg / mL to about 0.5 mg / mL.

41. A composition according to any one of claims 32 to 40, wherein the effective load comprises an RNA molecule.

42. The composition of claim 41, wherein the RNA molecule comprises more than one mRNA molecule.

43. The composition of claim 41, wherein the RNA molecule comprises mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or a combination thereof.

44. The composition of claim 43, wherein the RNA molecule comprises a sgRNA molecule.

45. The composition of claim 43, wherein the RNA molecules comprise sgRNA molecules and mRNA molecules.

46. ​​The composition of any one of claims 30 to 45, wherein the payload further comprises a protein.

47. A composition according to any one of claims 32 to 46, wherein the nucleic acid encodes an immunogen.

48. The composition of claim 47, wherein the nucleic acid encodes hemagglutinin (HA) or ovalbumin.

49. The composition of any one of claims 30 to 48, wherein the ionizable lipid comprises a lipid according to any one of Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V), or a combination thereof.

50. The composition of any one of claims 30 to 49, wherein the ionizable lipid comprises a lipid according to Formula (IA) or Formula (IB) or a combination thereof.

51. The composition of any one of claims 30 to 49, wherein the ionizable lipid comprises a lipid according to Formula (IIA), Formula (IIB) or Formula (IIC) or a combination thereof.

52. according to the composition of any one of claims 36 to 51, wherein the neutral lipid comprises cholesterol, sterol, 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 (POPC), palmitoylphosphatidylcholine (DPhPE), 1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPhPE), 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine (1-acyl-3 ... acyloleyl-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 (trans-DOPE) or a combination thereof.

53. A composition according to any one of claims 30 to 52, wherein the composition comprises a population of lipid nanoparticles.

54. The composition of claim 53, wherein the lipid nanoparticles have a diameter of about 20 nm to about 1 μm.

55. A composition according to any one of claims 30 to 52, wherein the composition comprises a liposome population.

56. The composition of any one of claims 30 to 55, wherein 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.

57. A method for targeting a payload to an immune cell of a subject, the method comprising administering to the subject a composition according to any one of claims 30 to 56.

58. The method of claim 57, wherein the immune cells comprise T cells, B cells, dendritic cells (DC), T helper cells, cytotoxic T cells (CTL), natural killer cells (NK), macrophages, or a combination thereof.

59. The method of claim 57, wherein the immune cells comprise spleen immune cells.

60. The method of claim 59, wherein the immune cells comprise dendritic cells.

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

62. The method of claim 61, wherein the lipid solution of (b) further comprises at least one neutral lipid.

63. A method for preparing a population of lipid formulations containing a payload molecule, the method comprising: (a) contacting a peptide comprising LLELLESL (SEQ ID NO: 1) with a lipid phase, wherein the lipid phase comprises 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 the lipid formulation complexed with the payload molecule.

64. The method of claim 63, wherein the lipid phase of (a) further comprises at least one neutral lipid.

65. The method of any one of claims 61 to 64, wherein the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).

66. The method of any one of claims 61 to 65, wherein the payload is a nucleic acid molecule.

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