[go: up one dir, main page]

EP4547282A1 - Compositions lipidiques pour administration in vivo - Google Patents

Compositions lipidiques pour administration in vivo

Info

Publication number
EP4547282A1
EP4547282A1 EP23750861.9A EP23750861A EP4547282A1 EP 4547282 A1 EP4547282 A1 EP 4547282A1 EP 23750861 A EP23750861 A EP 23750861A EP 4547282 A1 EP4547282 A1 EP 4547282A1
Authority
EP
European Patent Office
Prior art keywords
lipid
peptide
composition
payload
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23750861.9A
Other languages
German (de)
English (en)
Inventor
Evgenia VEROVSKAYA
Joel Jessee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Life Technologies Corp
Original Assignee
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Life Technologies Corp filed Critical Life Technologies Corp
Publication of EP4547282A1 publication Critical patent/EP4547282A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • compositions and methods which address this and other needs in the art.
  • methods for delivering a payload to a spleen cell in a subject including: (i) providing a lipid complex comprising at least one ionizable lipid, at least one peptide where the peptide comprises LLELLESL (SEQ ID NO: 1), and at least one payload molecule; and (ii) administering the lipid complex to a subject.
  • the lipid complex further comprises at least one neutral lipid.
  • the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24.
  • the lipid complex further includes a second peptide which comprises at least one of SEQ ID NO: 25 and/or SEQ ID NO: 26.
  • the peptide is at a concentration from about 0.001 to about 0.5 mg/mL, or from about 0.05 mg/mL to about 0.5 mg/mL. In some embodiments, the peptide is at a concentration from about 0.001 to about 0.5 mg/mL. In some embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.5 mg/mL.
  • the peptide is at a concentration of about 0.001 mg/mL. In other embodiments, the peptide is at a concentration of about 0.05 mg/mL. In other embodiments, the peptide is at a concentration of about 0.5 mg/mL.
  • the payload is a nucleic acid. In some embodiments, the at least one ionizable lipid comprises a charge (N), the payload nucleic acid comprises a charge (P), and the lipid complex comprises an N/P ratio from 0.01 to 0.2, or from 0.05 to 0.5, or from 0.1 to 1.0, or from 0.5 to 2.0, or from 1.0 to 5.0.
  • 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.
  • the N/P ratio is about 2.0.
  • the payload comprises an RNA molecule.
  • the RNA molecule includes mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or combinations thereof.
  • the RNA molecule includes more than one mRNA molecule.
  • the RNA molecule includes at least two mRNA molecules.
  • the RNA molecule includes an sgRNA molecule and an mRNA molecule.
  • the RNA molecule includes an sgRNA molecule.
  • the payload further includes a protein.
  • the nucleic acid payload encodes an immunogen.
  • the nucleic acid payload encodes for hemagglutinin (HA) or for ovalbumin.
  • the ionizable lipid includes a lipid according to Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V), or combinations thereof.
  • the ionizable lipid includes at least one lipid according to Formula (I). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (II).
  • the ionizable lipid includes at least one lipid according to Formula (III). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (IV). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (V). [0014] In some embodiments, the ionizable lipid includes a lipid according to Formula (IA) or Formula (IB), or combinations thereof. In some embodiments, the ionizable lipid includes a lipid according to Formula (IA). In some embodiments, the ionizable lipid includes a lipid according to Formula (IB).
  • the at least one ionizable lipid includes a lipid according to Formula (IA) and an ionizable lipid according to Formula (IB).
  • the ionizable lipid includes a lipid according to Formula (IIA), Formula (IIB), or Formula (IIC), or combinations thereof.
  • the ionizable lipid includes a lipid according to Formula (IIA).
  • the ionizable lipid includes a lipid according to Formula (IIB).
  • the ionizable lipid includes a lipid according to Formula (IIC).
  • the lipid complex includes liposomes. In some embodiments, the lipid complex includes lipid nanoparticles. In some embodiments, the lipid complex includes a lipid nanoparticle population, wherein the nanoparticle has a diameter from about 20 nm to about 1 ⁇ m. In some embodiments, the nanoparticle has a diameter from about 10 nm to about 900 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 800 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 700 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 600 nm.
  • the local administration comprises intramuscular administration or subcutaneous administration.
  • the administering comprises intravenous administration.
  • the administering comprises administration to the brain, spinal cord, eye or lymph node of a subject.
  • the subject includes a mammalian subject.
  • the subject includes a human.
  • the payload is delivered to a dendritic cell of the spleen.
  • the lipid complex targets an spleen cell of the subject about 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.5x or more compared to targeting with administration of a lipid complex comprising the ionizable lipid and the payload without the peptide.
  • Provided herein are methods for expressing a protein in spleen tissue in a subject, including administering the lipid complex described herein to the subject via systemic administration.
  • compositions for delivery of a payload to a spleen cell are provided herein.
  • compositions including: at least one ionizable lipid having a charge (N), at least one endosomal release 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.
  • the composition further comprises at least one neutral lipid.
  • the peptide has at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 2-24.
  • the composition further includes a second peptide which comprises at least one of SEQ ID NO: 25 and/or SEQ ID NO: 26.
  • the peptide is at a concentration from about 0.001 to about 0.5 mg/mL, or from about 0.05 mg/mL to about 0.5 mg/mL. In some embodiments, the peptide is at a concentration from about 0.001 to about 0.5 mg/mL. In some embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.5 mg/mL. In some embodiments, the peptide is at a concentration of about 0.001 mg/mL.
  • the peptide is at a concentration of about 0.05 mg/mL. In other embodiments, the peptide is at a concentration of about 0.5 mg/mL. [0027] In some embodiments, the N/P ratio is from about 0.01 to about 0.2. In some embodiments, the N/P ratio is from about 0.05 to about 0.5. In some embodiments, the N/P ratio is from about 0.1 to about 1.0. In some embodiments, the N/P ratio is from about 0.5 to about 2.0. In some embodiments, the N/P ratio is from about 1.0 to about 5.0. In some embodiments, the N/P ratio is less than about 0.1. In some embodiments, the N/P ratio is about 0.1.
  • 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.
  • the payload comprises an RNA molecule.
  • the RNA molecule includes mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, crRNA, tracrRNA, or combinations thereof. In some embodiments, the RNA molecule includes more than one mRNA molecule. In some embodiments, the RNA molecule includes at least two mRNA molecules.
  • the RNA molecule includes an sgRNA molecule and an mRNA molecule. In some embodiments, the RNA molecule includes an sgRNA molecule. [0029] In other embodiments, the payload further includes a protein. [0030] In some embodiments, the nucleic acid payload encodes an immunogen. In some embodiments, the nucleic acid payload encodes for hemagglutinin (HA) or for ovalbumin. [0031] In some embodiments, the ionizable lipid includes a lipid according to Formula (I), Formula (II), Formula (III), Formula (IV), or Formula (V), or combinations thereof. In some embodiments, the ionizable lipid includes at least one lipid according to Formula (I).
  • the ionizable lipid includes at least one lipid according to Formula (II). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (III). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (IV). In some embodiments, the ionizable lipid includes at least one lipid according to Formula (V). [0032] In some embodiments, the ionizable lipid includes a lipid according to Formula (IA) or Formula (IB), or combinations thereof. In some embodiments, the ionizable lipid includes a lipid according to Formula (IA). In some embodiments, the ionizable lipid includes a lipid according to Formula (IB).
  • the at least one ionizable lipid includes a lipid according to Formula (IA) and an ionizable lipid according to Formula (IB).
  • the ionizable lipid includes a lipid according to Formula (IIA), Formula (IIB), or Formula (IIC), or combinations thereof.
  • the ionizable lipid includes a lipid according to Formula (IIA).
  • the ionizable lipid includes a lipid according to Formula (IIB).
  • the ionizable lipid includes a lipid according to Formula (IIC).
  • the neutral lipid includes 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), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatid
  • DOPE dioleo
  • the composition includes liposomes. In some embodiments, the composition includes lipid nanoparticles. In some embodiments, the composition includes a lipid nanoparticle population, wherein the nanoparticle has a diameter from about 20 nm to about 1 ⁇ m. In some embodiments, the nanoparticle has a diameter from about 10 nm to about 900 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 800 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 700 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 600 nm.
  • the nanoparticle has a diameter from about 20 nm to about 500 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 400 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 300 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 200 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 100 nm. In some embodiments, the nanoparticle has a diameter from about 20 nm to about 50 nm.
  • 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.
  • methods of inducing an immune response in a subject including: administering to the subject the compositions described herein wherein the payload is an immunogen or encodes for an immunogen.
  • the administering includes systemic administration or local administration, wherein the local administration comprises intramuscular administration or subcutaneous administration.
  • the administering comprises intravenous administration.
  • the administering comprises administration to the brain, spinal cord, eye or lymph node of a subject.
  • the subject includes a mammalian subject.
  • the subject includes a human.
  • methods for delivering a payload to an immune cell of a subject where the method includes administering any of the compositions described herein to the subject via intravenous administration or via intramuscular administration.
  • methods for targeting a payload to an immune cell of a subject the method including administering the compositions described herein to the subject.
  • the immune cell includes T cell, B cell, dendritic cell (DC), T helper cell, cytotoxic T cell (CTL), natural killer cell (NK), macrophage, or combinations thereof.
  • the immune cell includes a spleen immune cell.
  • the immune cell includes a spleen dendritic cell.
  • the composition targets an immune cell of the subject about 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.5x or more compared to targeting with administration of a composition comprising the ionizable lipid and the payload without the peptide.
  • a method for preparing a population of lipid formulations containing a payload molecule including: (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 an ionizable lipid into the aqueous solution, wherein the injecting comprises extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (c) producing the population of lipid formulations complexed with the payload molecule.
  • Also provided herein is a method for preparing a population of lipid formulations containing a payload molecule, including: (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) producing the population of lipid formulations complexed with the payload molecule.
  • the lipid solution of (b) or the lipid phase of (a) further comprises at least one neutral lipid.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the payload molecule is a nucleic acid.
  • a kit including a composition having at least one ionizable lipid and at least one peptide where the peptide comprises LLELLESL (SEQ ID NO: 1).
  • the kit further includes at least one neutral lipid.
  • the ionizable lipid and neutral lipid are in a separate container from the peptide.
  • FIGS.1A-1C are graphs depicting in vivo luciferase flux localized to spleens 4 hours after intravenous administration of lipid-peptide formulations.
  • FIG.1A depicts results with different ionizable lipid formulations containing peptide.
  • FIGS.1B and 1C depict results of formulations with and without peptide.
  • FIG.2 is a graph depicting flux in spleens after intravenous administration of lipid-peptide formulations.
  • FIG.3 is a graph depicting flux in muscle after intramuscular administration of lipid-peptide formulations. The graph depicts results from formulations varying in N/P ratio from 0.1 to 5.0.
  • FIGs.4A-4B are data showing the effect of different N/P ratios (a ionizable lipid comprising a charge N and a nucleic acid molecule comprising a charge P) on biodistribution and delivery efficacy for formulations LP11 and LP12 following intravenous injection.
  • FIG.4A is a graph showing results from formulations varying in N/P ratio from 4.0 to 0.125..
  • FIG.4B are representative bioluminescence images showing the tissue specific delivery of the formulations with N/P 2 to the spleen of a mouse and not to the liver or lung. Scale bar is radiance (p/sec/cm2/sr).
  • FIG.5 is an image showing a schematic of using tdTomato reporter mice and Cre mRNA as lipid complex payload to analyze cell populations that are being targeted.
  • FIG.6 is a graph showing the specific delivery of the Cre mRNA containing LP11 formulation composition to the dendritic cell (DC) population in the spleen at administered doses of 5 ⁇ g, 10 ⁇ g and 40 ⁇ g Cre mRNA.
  • FIG.7A is a bar graph showing tdTomato expression in different splenocyte populations following intravenous administration of Cre mRNA-formulated lipid compositions using LP11, LP12, and the LP11 and LP12 formulations without SEQ ID NO:6 peptide (LP11 – no peptide and LP12 – no peptide).
  • FIG.7B are images showing representative gating in spleen dendritic cells (MHCII+/CD11C+ population) versus tdTomato expression following administration of Cre mRNA-formulated lipid compositions using LP11, LP12, LP11 – no peptide, and LP12 – no peptide.
  • FIGs.8A- 8B are data showing in vivo expression of fLuc mRNA-formulated LP11 formulation or LP11 formulation without SEQ ID NO:6 peptide (LP11 – no peptide) at different N/P ratios.
  • FIG.8A are representative in vivo bioluminescence images. Scale bar is radiance (p/sec/cm2/sr).
  • FIG.8B is a graph depicting the intensity of bioluminescence in the region of interest (ROI) used to calculate total flux (photons/second).
  • ROI region of interest
  • FIG.9 depicts an image of a schematic of lipid complex uptake analysis of formulations provided herein.
  • FIGs.10A-10B are data showing cellular uptake of mRNA complexed LP11 formulations or of mRNA complexed LP11 without SEQ ID NO:6 peptide formulations (LP11 – no peptide).
  • FIG.10A depicts representative fluorescent images of HEK293 cells at different time points after transfection (Red: Cy5 mRNA, blue: DAPI nucleus; scale bars show 10 ⁇ m.) In grayscale, the red is the lighter signal and the blue is the darker signal.
  • FIG.10B is a graph depicting quantitation of cellular uptake over time following transfection.
  • FIG.11 depicts an image of a schematic of lipid complex uptake and endosomal escape analysis of formulations provided herein.
  • FIG.12 depicts representative fluorescent images showing endosomal escape of mRNA complexed LP11 formulations or of mRNA complexed LP11 without SEQ ID NO:6 peptide formulations (LP11 – no peptide) in transfected HEK293 cells.
  • Red Cy5 mRNA (left two panels); Green: late endosomal marker (center two panels); Merged: Red Cy5 mRNA, Green late endosomal marker and Blue DAPI nucleus (right two panels); scale bars 10 ⁇ m.
  • FIG.13 depicts an image of a schematic for the immunogenicity study design.
  • FIGs.14A-14B are graphs showing the HA antigen-specific humoral immune response at 3 and 6 weeks.
  • FIG.14A is a graph showing the response via intramuscular (IM) delivery of the indicated mRNA doses at 0.01 mg/kg, 0.05 mg/kg, or 0.25 mg/kg.
  • FIG.14B is a graph showing the response via intravenous (IV) delivery of the indicated mRNA doses at 0.5 mg/kg or 1 mg/kg.
  • FIGs.15A-15C are data showing the HA antigen-specific cellular immune response.
  • FIG.15A is an exemplary image showing and ELISPOT assay.
  • FIG.15B is a graph showing IFN-gamma secretion in response to HA peptide stimulation from splenocytes after intramuscular delivery.
  • FIG.15C is a graph showing IFN-gamma secretion in response to HA peptide stimulation from splenocytes after intravenous delivery.
  • DETAILED DESCRIPTION [0062] We have developed compositions particularly effective for in vivo delivery of payload molecules to specific tissues and/or cell types, in particular to immune system tissues and cells.
  • the compositions include at least one ionizable lipid, an endosomal release peptide and a payload, and provide effective and efficient delivery of the payload specifically to the spleen following intravenous administration, particularly to dendritic cells of the spleen.
  • compositions provide effective and efficient delivery of the payload specifically to the injection site tissue, and to regional immune cells.
  • antigen-specific humoral and cellular immune responses were obtained following systemic or local administration of antigen-encoding mRNA complexed with the provided formulations.
  • exemplary payloads for delivery to immune system tissues and cells via the provided lipid complex compositions include nucleic acid molecules, protein molecules and/or other bioactive agents.
  • the payload for delivery to an immune cell is a therapeutic agent or a diagnostic agent.
  • the lipid complex compositions further include at least one neutral lipid.
  • a lipid composition which includes at least one ionizable lipid comprising a charge (N), at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO:1), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio from 0.01 to 0.2, or from 0.05 to 0.5, or from 0.1 to 1.0, or from 0.5 to 2.0, or from 1.0 to 5.0. In some embodiments, the N/P ratio may be less than about 0.1.
  • 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.
  • the lipid composition further comprises at least one neutral lipid.
  • the peptide comprises SEQ ID NO.1 and a polycationic nucleic acid binding domain.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • a composition for delivery of a payload to a spleen cell where the composition comprises at least one ionizable lipid and at least one peptide comprising the sequence LLELLESL (SEQ ID NO: 1).
  • the composition for delivery of a payload to a spleen cell further comprises at least one neutral lipid.
  • the peptide comprising SEQ ID NO.1 is present at a concentration of less than 1.0 mg/ml.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the composition for delivery of a payload to a spleen cell further comprises at least one payload.
  • a payload to an immune cell, such as a spleen cell in vitro, ex vivo, or in a subject.
  • an immune cell such as a spleen cell in vitro, ex vivo, or in a subject.
  • methods, compositions and kits for inducing an immune response in a subject are, inter alia, methods and compositions for making a population of lipid formulations containing a payload.
  • General Definitions [0070] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
  • 0.2-5 mg is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.
  • Compounds are generally described herein using standard nomenclature. For a recited compound having asymmetric center(s), all of the stereoisomers of the compound and mixtures thereof are encompassed unless otherwise specified. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. Where a recited compound exists in various tautomeric forms, the compound is intended to encompass all tautomeric forms. Certain compounds are described herein using general formulas that include variables (e.g., X, L1, L2, L3, Y, etc.).
  • each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. If moieties are described as being “independently” selected from a group, each moiety is selected independently from the other. Each moiety therefore can be identical to or different from the other moiety or moieties.
  • the number of carbon atoms in a hydrocarbyl moiety can be indicated by the prefix “Cx-Cy,” where x is the minimum and y is the maximum number of carbon atoms in the moiety.
  • C 1 -C 6 alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms.
  • C3-C6 cycloalkyl means a saturated hydrocarbyl ring containing from 3 to 6 carbon ring atoms.
  • a prefix attached to a multiple-component substituent only applies to the first component that immediately follows the prefix.
  • the term "carbocyclylalkyl” contains two components: carbocyclyl and alkyl.
  • C3-C6 carbocyclyl C1-C6 alkyl refers to a C3-C6 carbocyclyl appended to the parent molecular moiety through a C1-C6 alkyl group.
  • linking element links two other elements in a depicted chemical structure
  • the leftmost-described component of the linking element is bound to the left element in the depicted structure
  • the rightmost-described component of the linking element is bound to the right element in the depicted structure.
  • the chemical structure is -LS-M-LS''- and M is -N(RB)S(O)-
  • the chemical structure is -LS-N(RB)S(O)-LS''- .
  • a linking element in a depicted structure is a bond, then the element left to the linking element is joined directly to the element right to the linking element via a covalent bond.
  • a chemical structure is depicted as -L S -M-L S ' and M is selected as bond, then the chemical structure will be -L S -L S ''-. If two or more adjacent linking elements in a depicted structure are bonds, then the element left to these linking elements is joined directly to the element right to these linking elements via a covalent bond. For instance, if a chemical structure is depicted as -L S -M-L S ''-M'-L S ''-, and M and L S ' are selected as bonds, then the chemical structure will be -LS-M'-LS''-.
  • a moiety is described as being optionally substituted with up to a particular number of non-hydrogen radicals that moiety may be either unsubstituted, or substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the moiety, whichever is less.
  • any heterocycle with less than three substitutable positions will be optionally substituted by up to only as many non-hydrogen radicals as the heterocycle has substitutable positions.
  • tetrazolyl which has only one substitutable position
  • an amino nitrogen is described as being optionally substituted with up to two non-hydrogen radicals, then a primary amino nitrogen will be optionally substituted with up to two non-hydrogen radicals, whereas a secondary amino nitrogen will be optionally substituted with up to only one non-hydrogen radical.
  • alkenyl means a straight or branched hydrocarbyl chain containing one or more double bonds. Each carbon-carbon double bond may have either E (cis) or Z (trans) geometry within the alkenyl moiety, relative to groups substituted on the double bond carbons.
  • alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E- and Z,Z-hexadienyl and the like.
  • alkenylene refers to a divalent unsaturated hydrocarbyl chain which may be linear or branched and which has at least one carbon-carbon double bond.
  • alkyl means a straight or branched saturated hydrocarbyl chain.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, iso-amyl, and hexyl.
  • alkylene denotes a divalent saturated hydrocarbyl chain which may be linear or branched.
  • alkylene examples include, but are not limited to, -CH2-, - CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and -CH 2 CH(CH 3 )CH 2 -.
  • alkynyl means a straight or branched hydrocarbyl chain containing one or more triple bonds.
  • alkynyl include ethynyl, 1-propynyl, 2- propynyl, 3-propynyl, decynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
  • alkynyl refers to a straight-chain or branched-chain hydrocarbon radical having one or more triple bonds containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 10 carbon atoms.
  • alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl and the like.
  • alkoxy refers to an alkyl ether radical, wherein the term “alkyl” is defined above.
  • alkyl ether radicals examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.
  • 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 from 6-15 carbon atoms (i.e. (C 6-15 )aryl), and in another embodiment from 6-10 carbon atoms (i.e.
  • (C6-10)aryl optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen, (for example chloro), amino, carboxylate and hydroxy.
  • substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen, (for example chloro), amino, carboxylate and hydroxy.
  • aryl radicals include, but are not limited to phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.
  • aralkyl alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl and the like.
  • aralkoxycarbonyl alone or in combination, means a radical of the formula -C(O)-O-aralkyl in which the term “aralkyl” has the significance given above.
  • An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.
  • aryloxy alone or in combination, means a radical of the formula aryl- O- in which the term "aryl” has the significance given above.
  • alkynylene refers to a divalent unsaturated hydrocarbon group which may be linear or branched and which has at least one carbon-carbon triple bonds.
  • Representative alkynylene groups include, by way of example, C C , C C CH2 , C C CH2 CH2 , CH2 C ⁇ C-CH2-, -C ⁇ C-CH(CH3)-, and -CH2-C ⁇ C-CH(CH2CH3)-.
  • alkanoyl alone or in combination, means an acyl radical derived from an alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4- methylvaleryl, and the like.
  • aryloxyalkanoyl means an acyl radical of the formula aryl-O-alkanoyl wherein aryl and alkanoyl have the significance given above.
  • aralkanoyl means an acyl radical derived from an aryl-substituted alkanecarboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4- phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4- methoxyhydrocinnamoyl, and the like.
  • aroyl means an acyl radical derived from an aromatic carboxylic acid.
  • radicals include aromatic carboxylic acids, an optionally substituted benzoic or naphthoic acid such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4- benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6- (benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3- (benzyloxyformamido)-2-naphthoyl, and the like.
  • aminocarbonyl alone or in combination, means an amino-substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group continuing substituents selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals and the like.
  • aminoalkanoyl means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from the group consisting of hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N- dimethylaminoacetyl and N-benzylaminoacetyl.
  • carbocycle or “carbocyclic” or “carbocyclyl” refers to a saturated (e.g., “cycloalkyl"), partially saturated (e.g., “cycloalkenyl” or “cycloalkynyl") or completely unsaturated (e.g., "aryl”) 3- to 8-membered carbon ring system containing zero heteroatom ring atom.
  • Ring atoms or “ring members” are the atoms bound together to form the ring or rings.
  • a carbocyclyl may be, without limitation, a single ring, two fused rings, or bridged or spiro rings.
  • a substituted carbocyclyl may have either cis or trans geometry.
  • carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, decalinyl, and norpinanyl.
  • a carbocycle group can be attached to the parent molecular moiety through any substitutable carbon ring atom.
  • a carbocycle group is a divalent moiety linking two other elements in a depicted chemical structure
  • the carbocycle group can be attached to the two other elements through any two substitutable ring atoms.
  • a carbocycle group is a trivalent moiety linking three other elements in a depicted chemical structure
  • the carbocycle group can be attached to the three other elements through any three substitutable ring atoms, respectively.
  • the carbocycle may be attached at any endocyclic carbon atom which results in a stable structure.
  • Carbocycles in one embodiment have 5-7 carbons.
  • cycloalkyl refers to a saturated carbocyclyl group containing zero heteroatom ring member.
  • Non-limiting examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl and norpinanyl.
  • cycloalkyl alone or in combination, means an alkyl radical which contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • cycloalkylalkyl means an alkyl radical as defined above which is substituted by a cycloalkyl radical containing from about 3 to about 8, in one embodiment from about 3 to about 6, carbon atoms.
  • cycloalkylcarbonyl means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, and the like, or from a benz-fused monocyclic cycloalkanecarboxylic acid which is optionally substituted by, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2- naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.
  • cycloalkylalkoxycarbonyl means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.
  • carbocyclylalkyl refers to a carbocyclyl group appended to the parent molecular moiety through an alkylene group.
  • C3-C6carbocyclylC1-C6alkyl refers to a C 3 -C 6 carbocyclyl group appended to the parent molecular moiety through C 1 -C 6 alkylene.
  • cycloalkenyl refers to a non-aromatic, partially unsaturated carbocyclyl moiety having zero heteroatom ring member.
  • Representative examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, and octahydronaphthalenyl.
  • halo indicates that the substituent to which the prefix is attached is substituted with one or more independently selected halogen radicals.
  • C 1 - C 6 haloalkyl means a C 1 -C 6 alkyl substituent wherein one or more hydrogen atoms are replaced with independently selected halogen radicals.
  • Non-limiting examples of C1-C6haloalkyl include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, and 1,1,1- trifluoroethyl. It should be recognized that if a substituent is substituted by more than one halogen radical, those halogen radicals may be identical or different (unless otherwise stated).
  • a heterocyclyl may be, without limitation, a monocycle which contains a single ring.
  • monocycles include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl
  • fused-ring heterocycles include benzo-fused heterocyclyls, such as indolyl, isoindolyl, indoleninyl (also known as “pseudoindolyl”), isoindazolyl (also known as “benzpyrazolyl” or indazolyl), benzazinyl (including quinolinyl (also known as “1-benzazinyl”) and isoquinolinyl (also known as “2-benzazinyl”)), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including cinnolinyl (also known as “1,2-benzodiazinyl”) and quinazolinyl (also known as "1,3-benzodiazinyl”)), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl, benzo
  • a heterocyclyl may also be, without limitation, a spiro ring system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decanyl.
  • a heterocyclyl may comprise one or more sulfur atoms as ring members; and in some cases, the sulfur atom(s) is oxidized to SO or SO2.
  • the nitrogen heteroatom(s) in a heterocyclyl may or may not be quaternized, and may or may not be oxidized to N-oxide. In addition, the nitrogen heteroatom(s) may or may not be N-protected.
  • a heterocycle or carbocycle may be further substituted.
  • substituted refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO 2 , -N 3 , -CN, -NH 2 , protected amino, oxo, thioxo, -NH-C 2 -C 8 -alkenyl, - NH-C2-C8-alkynyl, -NH-C3-C12-cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, - dialkylamino, -diarylamino, -diheteroarylamino, -O-C 1 -C 12 -alkyl, -O-C 2 -C 8 -alkenyl, alkynyl,
  • N-protecting group or “N-protected” refers to those groups capable of protecting an amino group against undesirable reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3 rd ed., John Wiley & Sons, NY (1999)).
  • N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, or 4- nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfenyl groups such as phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl (trityl-S-); sulfinyl groups such as p- methylphenylsulfinyl (p-methylphenyl-S(O)-
  • N- protecting groups include formyl, acetyl, benzoyl, pivaloyl, t butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
  • halogen means fluorine, chlorine, bromine or iodine.
  • biologically active agent generally refers to a composition, complex, compound or molecule which has a biological effect or that modifies, causes, promotes, enhances, blocks or reduces a biological effect, or that enhances or limits the production or activity of, reacts with and/or binds to a second molecules which has a biological effect.
  • the second molecule can, but need not be, an endogenous molecule (e.g., a molecule, such as a protein or nucleic acid, normally present in the target cell).
  • a biological effect may be, but is not limited to, one that stimulates or causes an immunoreactive response; one that impacts a biological process in a cell, tissue or organism (e.g., in an animal); one that imparts a biological process in a pathogen or parasite; one that generated or causes to be generated a detectable signal; one that regulates the expression of a protein or polypeptide; one that stops or inhibits the expression of a protein or polypeptide; or one that causes or enhances the expression of a protein or polypeptide.
  • Biologically active compositions, complexes, compounds or molecules may be used in investigative, therapeutic, prophylactic and diagnostic methods and compositions and generally act to cause.
  • transfection enhancer or “transfection enhancing agent” as used herein refers to a compound when added to a transfection agent increases the efficiency of transfection (i.e., increases the percent of cells transfected), increases the level of expression of a transfection agent, or reduces the requirement for the amount of payload, for example nucleic acid or protein, required to give a biological response, or any combination of the enhancements above.
  • the transfection enhancer also helps deliver molecules that help downregulate expression such as siRNA, LNA’s and the like.
  • the transfection enhancing agent is a peptide including, for example, a cell surface ligand, a fusion agent, and/or a nuclear localization agent such as a nuclear receptor ligand peptide.
  • Transfection enhancing agents include the peptides and polypeptides described, for example, in Table 1 of U.S. Patent No.10,538,784, the contents of which is hereby incorporated by reference in its entirety.
  • the term “nucleic acid binding moiety” as used herein refers to a compound or molecule capable binding to nucleic acid.
  • the binding molecule is capable of noncovalently binding to nucleic acid, while in other embodiments, the binding molecule links covalently to a transfection enhaner, a cell surface ligand, a nuclear localization sequence, and/or a fusion agent.
  • the binding molecule can include but is not limited to spermine, spermine derivative, spermidine, histones or fragments thereof, protamines or fragments thereof, HMG proteins or fragments thereof, poly-lysine, poly-arginine, poly-histidine, polyamines and cationic peptides, nucleic acid intercalaters, protein nucleic acid sequences or aptamers.
  • polycationic nucleic acid binding moiety refers to a moiety containing multiple positive charges at physiological pH that allow the moiety to bind a negatively charged nucleic acid.
  • a polycationic nucleic acid binding moiety may be linked to, for example, a transfection enhancer such as a cell surface ligand, a fusion agent, and/or a nuclear localization peptide. The linkage may be covalent.
  • Suitable polycationic nucleic acid binding moieties include polyamines and polybasic peptides containing, for example, multiple lysine, arginine, ornithine, or histidine residues, such as between about 8-20 such residues.
  • Non limiting examples are the cationic peptides that are repeats of lysine or arginine, for example a sequence having between 8-20 lysine residues or between 8-20 arginine residues.
  • the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids.
  • a “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.
  • lysis agent or “endosomal release agent” as used herein refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane or cell membrane and freeing the transfection agent, such as payload transporter including an RNA or DNA transporter, into the cytoplasm of the cell.
  • lysis peptide refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.
  • lysis agents/endosomal release agents include choroquine, 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 (2016).
  • surface ligand or "cell surface ligand” refers to a chemical compound or structure which will bind to a surface receptor of a cell.
  • cell surface receptor refers to a specific chemical grouping on the surface of a cell to which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor facilitates the internalization of the ligand and attached molecules.
  • a cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosylphosphatidylinositol (GPI)- anchored proteins (e.g., ⁇ -adrenergic receptor, T-cell activating protein, Thy-1 protein, GPI- anchored 5' nucleotidase).
  • GPI glycosylphosphatidylinositol
  • a “receptor” is a molecule to which a ligand binds specifically and with relatively high affinity.
  • a receptor is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx.
  • epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis.
  • surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).
  • ligand refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptides and the like.
  • the ligand may also be a plant virus movement protein or peptide derived from such a protein.
  • Suitable peptides and proteins are described, for example, in US Patent No.10,538,784, the contents of which are hereby incorporated by reference in their entirety.
  • a ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides one method of targeting a payload, such as a polypeptide or a nucleic acid molecule, to specific cell types, depending on which cell surface ligand is used. Thus, use of a cell surface ligand may depend on the targeted cell type.
  • 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.
  • the lipid compositions further comprise at least one neutral lipid.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 1.
  • the peptide comprises a sequence selected from the group consisting of SEQ ID NOS: 1-24.
  • Exemplary payloads for delivery to tissues and cells via the provided lipid complex compositions include nucleic acid molecules, protein molecules and/or other bioactive agents.
  • the payload for delivery to an immune cell is a therapeutic agent or a diagnostic agent.
  • the payload such as one or more nucleic acids (e.g. mRNAs, siRNAs, sgRNAs), lipids, and amounts thereof may be selected to provide a specific N/P ratio.
  • the N/P ratio of the composition refers to the molar ratio of ionizable (in physiological pH) nitrogen atoms in one or more lipids to the number of phosphate groups in a nucleic acid (e.g., an RNA).
  • RNA- lipid nanoparticle N/P ratios eg, mRNA and siRNA payloads
  • lipid compositions comprising at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio of 0.01, or of 0.02, or of 0.04, or of 0.06, or of 0.08, or of 0.10, or of 0.12, or of 0.14, or of 0.16, or of 0.18, or of 0.20.
  • lipid compositions comprising at least one ionizable lipid having a charge (N), at least one neutral lipid, at least one peptide, wherein the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio of 0.01, or of 0.02, or of 0.04, or of 0.06, or of 0.08, or of 0.10, or of 0.12, or of 0.14, or of 0.16, or of 0.18, or of 0.20.
  • the N/P ratio is from 0.01 to 0.10.
  • the N/P ratio is from 0.01 to 0.20.
  • the N/P ratio is from 0.01 to 0.25. In other examples, the N/P ratio is from 0.01 to 0.33. In other examples, the N/P ratio is from 0.01 to 0.5. In other examples, the N/P ratio is from 0.01 to 1. In other examples, the N/P ratio is from 0.05 to 0.1. In other examples, the N/P ratio is from 0.05 to 0.125. In other examples, the N/P ratio is from 0.5 to 0.15. In other examples, the N/P ratio is from 0.05 to 0.167. In other examples, the N/P ratio is from 0.05 to 0.20. In other examples, the N/P ratio is from 0.05 to 0.25. In other examples, the N/P ratio is from 0.05 to 0.33.
  • the N/P ratio is from 0.05 to 0.5. In other examples, the N/P ratio is from 0.05 to 1.0. In some embodiments, the N/P ratio 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. [0128] In certain embodiments, the one or more nucleic acids (e.g. mRNAs, siRNAs, sgRNAs), lipids, and amounts thereof may be selected to provide an N/P ratio from about 2.0 to about 8.0, such as 2, 3, 4, 5, 6, 7, and 8.
  • the one or more nucleic acids e.g. mRNAs, siRNAs, sgRNAs
  • the N/P ratio may be from about 2.0 to about 5.0. In some embodiments, the N/P ratio may be about 4.0. In other embodiments, the N/P ratio is from about 5 to about 8. For example, the N/P ratio may be about 5.0, about 5.5, about 5.67, about 6.0, about 6.5, or about 7.0.
  • the one or more nucleic acids, lipids, and amounts thereof may be selected to provide an N/P ratio from 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 may be from about 5 to about 10.
  • the N/P ratio is from about 5 to about 20. In other embodiments, the N/P ratio may be from 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.
  • the ionizable lipid and peptide compositions provided herein encompass complexes in the form of lipid nanoparticles, liposomes (e.g., lipid vesicles) and lipoplexes.
  • liposome encompasses any compartment enclosed by a lipid bilayer.
  • liposome includes unilamellar vesicles which are comprised of a single lipid bilayer and generally have a diameter in the range of about 20 to about 400 nm. Liposomes can also be multilamellar having a diameter in the range of approximately 1 ⁇ m to approximately 10 ⁇ m. Multilamellar liposomes may consist of several (anywhere from two to hundreds) unilamellar vesicles forming one inside the other in diminishing size, creating a multilamellar structure of concentric phospholipid spheres separated by layers of water. Alternatively, multilamellar liposomes may consist of many smaller nonconcentric spheres of lipid inside a large liposome.
  • liposomes include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV).
  • the compositions include liposomes which contain any suitable ionizable lipid and neutral lipids, along with the peptide as provided herein.
  • the compositions include lipid nanoparticles (LNPs). LNP composition are typically sized on the order of micrometers or small and may include a lipid bilayer.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 1 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 900 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 800 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 700 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 600 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 500 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 400 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 300 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 200 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 100 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 20 nm to about 50 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 900 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 800 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 700 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 600 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 500 ⁇ m.
  • the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 400 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 300 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 200 ⁇ m. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is from about 100 nm to about 150 ⁇ m. [0132] The characteristics of a lipid nanoparticle (e.g., an LNP without payload or an LNP with payload) may depend on the components thereof.
  • a lipid nanoparticle including cholesterol as a structural lipid may have different characteristics than a lipid nanoparticle that includes a different structural lipid.
  • the characteristics of a lipid nanoparticle may depend on the absolute or relative amounts of its components. For instance, a lipid nanoparticle including a higher molar fraction of a phospholipid may have different characteristics than a lipid nanoparticle including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. [0133] Lipid nanoparticles may be characterized by a variety of methods.
  • microscopy e.g., transmission electron microscopy or scanning electron microscopy
  • Dynamic light scattering or potentiometry e.g., potentiometric titrations
  • Dynamic light scattering may also be utilized to determine particle sizes.
  • Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.
  • the lipid complex compositions provided herein may be relatively homogenous.
  • a polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the lipid nanoparticles.
  • a small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution.
  • a lipid nanoparticle may have a polydispersity index from 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.
  • the polydispersity index of a lipid nanoparticle may be from about 0.10 to about 0.20.
  • the zeta potential of a lipid nanoparticle may be used to indicate the electrokinetic potential of the composition.
  • the zeta potential may describe the surface charge of a nanoparticle composition. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body.
  • the zeta potential of a lipid nanoparticle may be from about ⁇ 10 mV to about +20 mV, from about ⁇ 10 mV to about +15 mV, from about ⁇ 10 mV to about +10 mV, from about ⁇ 10 mV to about +5 mV, from about ⁇ 10 mV to about 0 mV, from about ⁇ 10 mV to about ⁇ 5 mV, from about ⁇ 5 mV to about +20 mV, from about ⁇ 5 mV to about +15 mV, from about ⁇ 5 mV to about +10 mV, from about ⁇ 5 mV to about +5 mV, from about ⁇ 5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +10 mV, from
  • the payload of the lipid complex composition is an RNA molecule.
  • the payload RNA molecule comprises mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, or combinations thereof.
  • 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).
  • the payload includes at least one sgRNA.
  • the payload of the lipid complex composition molecule includes an sgRNA molecule and an mRNA molecule.
  • 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).
  • 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.
  • the RNA molecule includes sgRNA, a crRNA, and/or a tracrRNA.
  • the lipid complex composition payload includes an RNP and an sgRNA.
  • the RNP can include a Cas protein and a sgRNA, a crRNA or a tracrRNA.
  • the RNA may encode a gene editing protein (e.g., an RNA encoding a ZFN, TALEN, Cas protein, Cre recombinase, etc).
  • the lipid complex composition payload includes an RNA encoding a gene editing protein and an sgRNA.
  • the lipid complex composition payload can include an RNA encoding a Cas protein and a sgRNA, a crRNA or a tracrRNA.
  • the nucleic acid payload of the lipid complex compositions is a single-stranded molecule.
  • the payload may include donor DNA.
  • the DNA payload may be a plasmid DNA or linear DNA.
  • the payload may be an RNP and include a Cas protein and a sgRNA, a crRNA or a tracrRNA.
  • the gene editing payload induces single-strand or double- strand breaks in DNA within the cells.
  • the gene editing reagent further comprises a repair template polynucleotide.
  • the repair template comprises (a) a first flanking region comprising nucleotides in a sequence complementary to about 40 to about 90 base pairs on one side of the single or double strand 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 or double strand break; or (b) a first flanking region comprising nucleotides in a sequence complementary to at least about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 base pairs on one side of the single or double strand 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 or double strand break.
  • Non-limiting descriptions relating to gene editing (including repair templates) using the CRISPR-Cas system are discussed in Ran et al. (2013) Nat Protoc.2013 Nov; 8(11): 2281-2308, the entire content of which is incorporated herein by reference. Embodiments involving repair templates are not limited to those comprising the CRISPR Cas system.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.
  • the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2 and in the NCBI database as under 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 a Streptococcus thermophilus Cas9 protein, the amino acid sequence of which may be found in the UniProt database under accession number Q03JI6.1.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme is Cas9, and may be Cas9 from S.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • nickases may be used for genome editing via homologous recombination.
  • the payload may include a Cas9 nickase used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.
  • the payload of the lipid composition includes an RNA molecule, and the RNA molecule includes sgRNA, a crRNA, a tracrRNA, or combinations thereof.
  • the payload of the lipid composition is an immunogen.
  • the nucleic acid payload of the lipid composition encodes for an immunogen.
  • the payload nucleic acid of the lipid composition encodes for a hemagglutinin (HA) protein or fragment thereof.
  • the nucleic acid payload of the lipid composition encodes for ovalbumin or fragment thereof.
  • the lipid composition delivers a payload which induces an immune response in a subject to the protein or nucleic acid-encoded protein of the lipid composition.
  • Exemplary transfection enhancing peptides for use in the lipid composition are provided herein.
  • such peptides comprise the sequence LLELLESL (SEQ ID NO: 1) and optionally comprise a polycationic nucleic acid binding moiety.
  • 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).
  • 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 moiety.
  • suitable polycationic nucleic acid binding moieties include without limitation polyamines and polybasic peptides containing, for example, poly-arginine, poly-lysine, poly-histidine, and/or poly-ornithine sequences with, for example, lengths of about 8 to about 20 residues.
  • the peptides for use in the compositions provided herein are at least 10 amino acids in length. In some embodiments, the peptides are at least 10 to about 100, at least 10 to about 75, at least 10 to about 50, at least 10 to about 40, at least 10 to about 30, or at least 10 to about 20 amino acids in length.
  • the peptides are about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 50, about 15 to about 60, or about 15 to about 70 amino acids in length. In some embodiments, the peptides are about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 70, or about 20 to about 80 amino acids in length. [0148] In some examples, the peptide has at least 80% sequence identity to SEQ ID NO: 6 (GLFEALLELLESLWELLLEA). In other examples, the peptide includes SEQ ID NO: 6, or fragments thereof. In other examples, the peptide has at least 85% sequence identity to SEQ ID NO: 6.
  • the peptide has at least 90% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 91% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 92% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 93% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 94% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 95% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 96% sequence identity to SEQ ID NO: 6. In other examples, the peptide has at least 97% sequence identity to SEQ ID NO: 6.
  • Polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, in which the remaining amino acid sequence is usually identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are 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.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of an entire polypeptide sequence or an individual domain thereof), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • a specified region e.g., of an entire polypeptide sequence or an individual domain thereof
  • two sequences are 100% identical. In embodiments, two sequences are 100% identical over the entire length of one of the sequences (e.g., the shorter of the two sequences where the sequences have different lengths).
  • identity may refer to the complement of a test sequence. In embodiments, the 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.
  • the 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.
  • Exemplary peptides useful in the formulations provided herein include, without limitation, the peptides of Table 1.
  • any of the SEQ ID NO: 1-22 peptides may further comprise arginine residues or additional arginine residues (e.g., R2, R4, R6, R8, R12) at the N or C terminus.
  • any of SEQ ID NO: 122 may further comprise lysine residues or additional lysine (e.g., K2, K4, K6, K8, K12) at the N or C terminus.
  • any of the SEQ ID NO: 1-22 peptides may comprise multiple histidine residues (e.g., H2, H4, H6, H8, H12) at the N or C terminus. In other embodiments, any of the SEQ ID NO: 1-22 peptides may comprise multiple ornithine residues at the N or C terminus. In certain embodiments, the SEQ ID NO: 1-22 sequence is connected to the multiple arginine, lysine, histidine or ornithine residues by a spacer or linker molecule.
  • formulations may include additional transfection enhancing agents such as a cell surface ligand peptide and/or a nuclear localization agent such as a nuclear receptor ligand peptide.
  • transfection enhancing agents include, but are not limited to, reovirus-related fusogenic peptides, insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein, a arginine rich cell permeability protein, a high mobility group protein, and invasin protein, and internalin protein, an endotoxin, a diphtheria toxin, a shigella toxin, a melittin, a magainin, a gramicidin, a cecrophin, a def
  • 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, without limitation, the exemplary peptides of Table 2.
  • SEQ ID NO: 25 amino acid sequence of PSYYRYD
  • PSYYRGD PSYYRGD
  • Table 2 SEQ ID NO: Sequence
  • the transfection enhancing peptide comprises at least two amino acids
  • the transfection enhancing peptide has at least 80% sequence identity to any one of SEQ ID NO: 27-36. In other examples, the transfection enhancing peptide includes any one of SEQ ID NO: 27-36, or fragments thereof. In other examples, the transfection enhancing peptide has at least 85% sequence identity to any one of SEQ ID NO: 27-36. In other examples, the transfection enhancing peptide has at least 90% sequence identity to any one of SEQ ID NO: 27- 36.
  • the transfection enhancing peptide has at least 95% sequence identity to any one of SEQ ID NO: 27-36. In other examples, the transfection enhancing peptide has 100% sequence identity to any one of SEQ ID NO: 27-36. In some embodiments, any of the SEQ ID NO: 27-36 peptides may further comprise additional arginine residues (e.g., R2, R4, R6, R8, R10, R12, R14, R16, R18, R20) at the N or C terminus.
  • additional arginine residues e.g., R2, R4, R6, R8, R10, R12, R14, R16, R18, R20
  • any of SEQ ID NO: 28, 30, 32, 34, and 36 may further comprise lysine residues (e.g., K2, K4, K6, K8, K10, K12, K14, K16, K18, K20) at the N or C terminus.
  • any of the SEQ ID NO: 27-36 peptides may comprise multiple histidine residues (e.g., H2, H4, H6, H8, H12) at the N or C terminus.
  • any of the SEQ ID NO: 27-36 peptides may comprise multiple ornithine residues at the N or C terminus.
  • the SEQ ID NO: 27- 36 sequence is connected to the multiple arginine, lysine, histidine or ornithine residues by a spacer or linker molecule.
  • the lipid compositions provided include a peptide that has comprises LLELLESL (SEQ ID NO: 1) and a peptide that comprises at least one or more sequences of PSYYRYD (SEQ ID NO: 25) and/or PSYYRGD (SEQ ID NO: 26).
  • the lipid compositions provided include a peptide that has at least 80% sequence identity to SEQ ID NO: 6 and a peptide that has at least 80% sequence identity to SEQ ID NO: 27.
  • the lipid compositions include a peptide that has at least 80% sequence identity to SEQ ID NO: 6 and a peptide that has at least 80% sequence identity to SEQ ID NO: 33.
  • the lipid compositions include a peptide selected from the peptides of SEQ ID NOs: 6-24 and a peptide selected from the peptides of SEQ ID NOs: 27-36.
  • the endosomal release peptides and/or cell surface ligand peptides for use in the provided compositions may be linked to a glycosylphosphatidylinositol (GPI) anchor peptide, such as for example, FTLTGLLGTLVTMGLLT (SEQ ID NO: 37).
  • GPI glycosylphosphatidylinositol
  • the peptides described herein are attached directly to the nucleic acid binding molecule by covalent bonding, or are connected to the binding molecule via a spacer.
  • spacer or “linker,” which are used interchangeably herein, as used herein refers to a chemical structure that links two molecules to each other.
  • the spacer binds each molecule on a different part of the spacer molecule.
  • the spacer is a hydrophilic moiety and comprises about 6 to 30 carbon atoms.
  • the spacer comprises a polyether, for example -CH2-O-(CH2-CH2-O-)iCH2-.
  • the spacer comprises a hydrophilic polymer, for example [(gly)i(ser)j]k.
  • i ranges from 1 to 6
  • j ranges from 1 to 6
  • k ranges from 3 to 20.
  • the spacer is a peptide of sequence APYKAWK (SEQ ID NO: 38).
  • the spacer is a sequence that is degraded in vivo by a peptidase.
  • the peptides for use in the compositions provided herein are at least 10 amino acids in length.
  • the peptides are at least 10 to about 100, at least 10 to about 75, at least 10 to about 50, at least 10 to about 40, at least 10 to about 30, or at least 10 to about 20 amino acids in length. In certain embodiments, the peptides are about 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 50, about 15 to about 60, or about 15 to about 70 amino acids in length. In some embodiments, the peptides are about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 60, about 20 to about 70, or about 20 to about 80 amino acids in length.
  • the lipid compositions provided include a peptide, wherein peptide is at a concentration from about 0.001 to about 0.5 mg/mL. In embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.5 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.001 to about 0.05 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.001 to about 0.1 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.01 to about 0.5 mg/mL.
  • the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.01 to about 0.4 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.01 to about 0.3 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.01 to about 0.2 mg/mL. In examples, the lipid composition includes a peptide, wherein peptide is at a concentration from about 0.01 to about 0.1 mg/mL. In embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.1 mg/mL.
  • the peptide is at a concentration from about 0.05 mg/mL to about 0.2 mg/mL. In embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.3 mg/mL. In embodiments, the peptide is at a concentration from about 0.05 mg/mL to about 0.4 mg/mL. In embodiments, the peptide is at a concentration from about 0.1 mg/mL to about 0.5 mg/mL. In embodiments, the peptide is at a concentration from about 0.1 mg/mL to about 0.4 mg/mL. In embodiments, the peptide is at a concentration from about 0.1 mg/mL to about 0.3 mg/mL.
  • the peptide is at a concentration from about 0.1 mg/mL to about 0.2 mg/mL.
  • ionizable lipid refers to a lipid having one or more functional groups that can reversibly be ionized (protonated or deprotonated) depending on the pH of the medium containing the lipid.
  • the functional group may be basic, such as an amino function, or may be acidic, such as a carboxylic acid moiety. The skilled artisan will be aware that other ionizable functional groups also may be used.
  • An ionizable lipid may contain both basic and acid moieties.
  • an ionizable lipid carries an overall positive charge at physiological pH.
  • Ionizable lipids described herein refer to lipids that have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH.
  • the ionizable lipids provided herein have a pKa of the protonatable group in the range of about 4 to about 11, e.g., about 4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and 6.9, when incorporated into the lipid compositions, for example, liposomes, lipid nanoparticles or other lipid complexes.
  • Ionizable lipids include, for example amine-containing lipids that can be readily protonated.
  • the ionizable lipid may be selected from, for example, the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-hydroxypropane- 1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N- 1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1- (2,3-diaoleoyloxypropyl)-2-(
  • ionizable lipids described in U.S. Patent 7,173,154, U.S. Patent No: 9,856,496, and U.S. Publication No: US 2019/0060482 are contemplated for use in the present compositions and methods (wherein the reference is incorporated by reference in their entireties).
  • lipid compositions can include at least a first ionizable lipid and at least a first neutral lipid, wherein said lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein said the ionizable lipids have the structure of Formula (I): (Formula (I)), and salts t ereo ; w ere: R1 and R2, independently, are an alkyl, alkenyl or alkynyl groups, having from 8 to 30 carbon atoms; an alkyl, alkenyl or alkynyl groups, having from 8 to 30 carbon atoms and optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, an ester, a mercaptan, alkylthio, or a carbamoyl group or where R1 is — (CH2)q— N(R)
  • a particularly preferred though non-limiting ionizable lipid used in the formation of complexes has the structure Formula IA: (Formula IA) and salts thereof, tituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 3 and R 4 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; m is an integer from 1 to 6; X a - is an anion
  • a particularly preferred though non-limiting ionizable lipid used in the formation of complexes has the structure Formula IB: 7 5 R R - b (Formula IB) and salts thereof, wherein R and R 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; R 6 and R 7 are independently substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer
  • R 1 and R 2 are independently substituted or unsubstituted alkyl. In embodiments, R 1 and R 2 are independently unsubstituted alkyl. R 1 and R 2 are independently unsubstituted C 1 -C 20 alkyl. In embodiments, R 1 and R 2 are independently unsubstituted C 5 -C 20 alkyl. In embodiments, R 1 and R 2 are independently unsubstituted C10-C20 alkyl. In embodiments, R 1 and R 2 are independently unsubstituted C12-C18 alkyl. In embodiments, R 1 and R 2 are independently unsubstituted C 14 -C 16 alkyl.
  • R 1 is unsubstituted C 14 alkyl.
  • R 2 is unsubstituted C14 alkyl.
  • R 1 is unsubstituted C15 alkyl.
  • R 2 is unsubstituted C15 alkyl.
  • R 1 is unsubstituted C16 alkyl.
  • R 2 is unsubstituted C 16 alkyl.
  • R 1 is –(CH 2 ) 13 CH 3 .
  • R 2 is –(CH 2 ) 13 CH 3 .
  • R 3 and R 4 are independently hydrogen or substituted or unsubstituted alkyl.
  • R 3 and R 4 are independently hydrogen.
  • R 5 , R 6 and R 7 are independently hydrogen, substituted or unsubstituted alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 1 -C 20 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 5 -C 20 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C10-C20 alkyl.
  • R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C12-C18 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 14 -C 16 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C14 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C15 alkyl. In embodiments, R 5 , R 6 and R 7 are independently hydrogen or unsubstituted C 16 alkyl. In embodiments, R 5 is unsubstituted C 14 alkyl. In embodiments, R 7 is unsubstituted C 14 alkyl.
  • R is (CH2)13CH3. In embodiments, R is hydrogen. In embodiments, R is (CH2)13CH3. [0172] In embodiments, R 8 is hydrogen or substituted or unsubstituted alkyl. In embodiments, R 8 is hydrogen. [0173] 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.
  • 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 5.
  • n is 6.
  • R 1 is –(CH 2 ) 13 CH 3
  • R 2 is –(CH 2 ) 13 CH 3
  • R 3 is hydrogen
  • R 4 is hydrogen
  • m is 4
  • Xa is CH3COO-.
  • R 5 is –(CH 2 ) 13 CH 3
  • R 6 is hydrogen
  • R 7 is –(CH 2 ) 13 CH 3
  • R 8 is hydrogen
  • n is 4
  • X b - is CH 3 COO-.
  • Examples of compounds of Formula I structure for use as an ionizable lipid in the provided lipid compositions and methods include, without limitation, the following: Compound 1-1: , Compo un - : , Compound 13: nd . [ ed in U.S. Patent No.9,259,475 and U.S. Patent No.10,883,118 are contemplated for use in the present compositions and methods (wherein the references are incorporated by reference in their entirety). In some embodiments, such lipids are based on a core of N,N’-disubstituted 2,3-hihydroxy-1,4- butanediamine.
  • the lipid composition comprises a ionizable diaminobutane lipid molecule having the structure of Formula II: (Formula II), and salts thereof, where dently is C 1 -C 23 alkyl, C 1 -C 23 alkenyl, —(CO)C 1 -C 23 alkyl, or —(CO)C 1 - C23 alkenyl; and each R 2 independently is —CH2—(CHR 3 )1-6—CH2—NHR 4 or —CH2— (CHR 3 )0-6—CH2—OH, where each R 3 independently is H, OH, or NH2, R 4 is H or CH3; and R 10 is H or C 1 -C 8 alkyl.
  • Formula II Formula II
  • R 1 may be C14-C20 alkyl or monounsaturated C14-C20-alkenyl.
  • R 2 may be —CH2—(CHR 3 )1-6—CH2—NHR 4 and R 3 is H or OH. In some embodiments, no more than 3 R 3 groups are OH in each R 3 moiety.
  • each R 1 may be the same or different, and each R 2 may be the same or different.
  • one or both R 10 may be H, or one or both R 10 may be C 1 -C 3 alkyl, for example, one or both R 10 may be methyl.
  • one or both R 1 may be C 12 -C 20 alkenyl, and the alkenyl moieties may be cis alkenyl.
  • one or both R may be CH2 CHOH (CHR )0-5 CH2—NHR 4 ; for example one or both R 2 may be —CH2—CHOH—CH2—NH2.
  • R advantageously is C 14-18 alkyl or C 14-18 monounsaturated alkenyl.
  • Each R 1 , R 2 and/or each R 10 may be the same or different and thus the molecule may be symmetrical or non-symmetrical.
  • R 2 may be C1-C3 alkyl and/or R 1 is monounsaturated C 12 -C 20 alkenyl.
  • One or both C 12 -C 20 alkenyl moieties in R 1 when present, may be cis alkenyl.
  • each R 1 independently may be C1-C23 alkyl, C1-C23 alkenyl, — (CO)C1-C23 alkyl, or —(CO)C1-C23 alkenyl.
  • Each R 2 independently may be C1-C6 alkyl, or C1- C 6 alkenyl, optionally interrupted by up to 2 O atoms.
  • Each R 3 independently may be H, C 1 - C 6 alkyl, C 1 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloalkyl-C 1 -C 6 alkyl, C 5 -C 7 - cycloalkenyl, C5-C7-cycloalkenyl-C1-C6 alkyl, —(CH2)mNR 6 (CH2)nNHR 7 , —(CH2)2-6NHR 7 , — (CH 2 ) 3-6 NHC( ⁇ NH)NH 2 , ((CH 2 ) m O x ) y (CH 2 ) z O x R 8 , or —(CH 2 ) 0-3 Het.
  • Each R 4 independently may be H, C 1 -C 6 alkyl, or C 1 -C 6 alkenyl and each R 5 independently may be H, an amine protecting group, —(CH2)mNR 6 (CH2)nNHR 7 , —(CH2)2-6NHR 7 , —(CH2)3-6NHC( ⁇ NH)NH2, — (CO)C1-C23 alkyl, —(CO)C1-C23 alkenyl, or a peptide containing 1-20 amino acid residues.
  • the peptide advantageously contains multiple positively charged amino acid side chains.
  • the peptide may contain one or more lysine, arginine, and/or histidine residues.
  • amino acids also may be used, whether or not naturally occurring.
  • ornithine, homo-arginine and other amino acids containing amine, guanidine; imidazole and other basic heterocycles and the like can be used.
  • Each m independently may be 2-5, and each n independently may be 2-5, while 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)C 1 -C 23 alkyl, or —(CO)C 1 -C 23 alkenyl
  • R 8 may be H, C 1 -C 6 alkyl, or C 1 -C 6 alkenyl
  • Het may be a 57 membered monocyclic basic heterocycle, or an 811 membered bicyclic basic heterocycle.
  • R 3 may be —(CH 2 ) m NR 6 (CH 2 ) n NHR 7 and/or R 5 may be —(CH2)2-6NHR 7 .
  • m may be 3
  • n may be 3
  • R 5 may be —(CH2)3NHR 7 .
  • each R 6 and each R 7 may be H.
  • at least one R 6 or R 7 may be —(CO)C 1 - C23 alkyl, or —(CO)C1-C23 alkenyl and the remainder are H.
  • each R 1 may be C12-C20 alkyl or C12-C20 monounsaturated alkenyl
  • each R 2 may be C1-C3 alkyl
  • each R 3 may be —(CH 2 ) 3 NH(CH 2 ) 3 NH 2
  • each R 4 may be H
  • each R 5 may be —(CH 2 ) 3 NH 2 .
  • each R 1 may be C 12 -C 20 alkyl or C 12 -C 20 monounsaturated alkenyl
  • each R 2 may be C1-C3 alkyl
  • each R 3 may be —(CH2)3NR 6 (CH2)3NHR 7
  • each R 4 may be H
  • each R 5 may be —(CH 2 ) 3 NHR 7
  • at least one R 6 or R 7 may be —(CO)C 1 -C 23 alkyl, or —(CO)C 1 - C 23 alkenyl and the remainder are H.
  • each R for example may be, but is not limited to, C12-20 alkyl or C12-20 alkenyl; and each R and each R′′ independently may be, but is not limited to, H, an amine protecting group, — (CO)C 1 -C 23 alkyl, or —(CO)C 1 -C 23 alkenyl.
  • R may be C14- C18 alkyl or C14-C18 alkenyl, and/or each R′ and each R′′ independently may be H, C14-C18 alkyl or C 14 -C 18 alkenyl.
  • Another specific example of the compounds of structure Formula II is the set of compounds having the structure: (Formula IIC), and salts thereof, , , be, but is not limited to, C12-20 alkenyl; and/or each R′ and each R′′ independently may be, but is not limited to, H, an amine protecting group, —(CO)C1-C23 alkyl, or —(CO)C 1 -C 23 alkenyl.
  • R may be oleyl and each R′ and each R′′ independently may be H, or oleoyl. In still other embodiments at least one R′ or R′′ may be oleoyl and the remainder are H.
  • each R 3 independently may be —(CH2)2-6NHR 7 , —(CH2)3-6NHC( ⁇ NH)NH2, or —(CH2)1-3Het, each R 4 is H and each R 5 independently is H or a peptide containing 1-20 amino acid residues.
  • each R 1 may be C 12 -C 20 alkyl or C 12 -C 20 alkenyl
  • each R 2 may be C1-C3 alkyl
  • each R 5 may be H.
  • each R 3 may be —(CH2)2-6NH2, —(CH2)3-6NHC( ⁇ NH)NH2 or each R 3 may be —(CH2)1-3Het. In one specific embodiment, Het may be .
  • a non limiting ionizable lipid used in the composition may be compounds having the following Formula II structures, where R is C 14 , C 16 , or C 18 alkyl, or C 14 , C 16 , or C 18 monounsaturated alkenyl: Compound 2-1: Compound 2-2: Compound 2-3: Compound 2-4:
  • the lipid compositions can include at least a first ionizable lipid and optionally at least a first neutral lipid, wherein said lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein said the ionizable lipids have the structure of Formula III: , .
  • X 1 and X 2 may independently be selected from the group consisting of (CH2)n, (CHOH)n, and CONH.
  • X5 and X6 independently may be (CH2)1-6.
  • W1 and W2 independently may be selected from the group consisting of, hydrogen, —OH, —O— (C 1 -C 30 )alkyl, —O—(C 1 -C 30 )alkenyl, —O—(C 1 -C 30 )alkynyl, —NH 2 , —NH(CH 2 ) s CH 3 , — N((CH2)sCH3), —SH, and —NH—NH2.
  • R4 and R5 independently may be selected from the group consisting of (CH 2 ) n , (CH 2 —CHOH—CH 2 ) n , (CHOH) n , HNCO, CONH, CO, —O—, —S—, —S—S—, polyamide and an ester linkage.
  • L 1 and L2 independently may be selected from the group consisting of —NH—, —O—, — NHCO , CONH , OCO , COO , CO , S , S S , NHC(O)O , OC(O)NH—, —NHCONH—, —NHC( ⁇ NH)NH—, —S(O)— and —SO2—.
  • Y is a heterocyclic moiety containing at least one amine or amide moiety.
  • the points of attachment of Y may be carbon and/or heteroatoms.
  • suitable heterocyclic moieties include, but are not limited to, piperazine, piperidine, pyridine, pyrrolidine, and imidazole moieties and derivatives thereof.
  • the heterocyclic moiety is a piperazine ring, where the points of attachment optionally are at one or both of the nitrogen atoms.
  • the heterocyclic moiety may optionally be substituted with up to 4 substituents independently selected from the group consisting of OH, ⁇ O, a carboxylic acid, an ether, a polyether, an alkylaryl, an amino alcohol, an amide, an straight chain alkyl, branched alkyl, cycloalkyl, straight chain alkenyl, branched alkenyl, cycloalkenyl, straight chain alkynyl, branched alkynyl, primary alkylamine, secondary alkylamine, tertiary alkyl amine, quaternary alkylamine, alkenylamine, secondary alkenylamine, tertiary alkenyl amine, quaternary alkenylamine, alkynylamine, secondary alkynylamine, tertiary alkynylamine, quaternary alkynylamine, amino alcohol, alcohol, ether, polyether, aryl, benzyl, heterocycle,
  • an alkylamine can be an amine containing a short or a long alkyl chain.
  • an alkenylamine will be understood to contain a short or long alkenyl chain, and the same is true for alkynylamines.
  • Z 1 and Z 2 independently may be selected from the group consisting of straight chain alkyl, branched alkyl, cycloalkyl, straight chain alkenyl, branched alkenyl, cycloalkenyl, straight chain alkynyl, branched alkynyl where m, n, p, and s independently are 06, with the proviso that when m, n, and p all are 0 then Y is eliminated and R3 is bonded directly to X2.
  • Y may have the following cyclic structure 4 may independently be selected from N and CH and n 1 and n 2 independently are 1-10.
  • Y is a 6-9 membered ring and, in exemplary specific embodiments, X3 and X4 are both N and n1 and n2 are both 2, i.e. Y is an optionally substituted piperazine moiety.
  • This structure may optionally be substituted with 1-4 moieties as described above for Y.
  • Y can have the following cyclic structure: , independently are 1-10. Typically, n1+n2 is 3-7. Such a cyclic structure may optionally be substituted with 1-4 moieties independently selected as described above for Y.
  • Examples of the lipids may be defined by the following structure, where L 1 and L2 both are NH and X5 and X6 are CH2: (Formula IIIA), and salts th .
  • R1-R6, W1, W2, X1, X2, Z1, Z2, Y, m, p, and q are as defined above.
  • a short chain alkyl group is typically, unless otherwise defined, C 1 -C 6 alkyl.
  • a long chain alkyl group is typically, unless otherwise defined, C10-C20 alkyl, or C10-C30 alkyl. When not specifically defined, either definition may be used, as appropriate.
  • other derivative groups containing alkyl moieties for example, alkoxy moieties and the like, also may contain short and/or long chain groups as appropriate in the context, unless otherwise defined.
  • An alkenyl group contains at least one cis or trans carbon-carbon double bond and typically is C10-C30 in chain length.
  • alkenyl groups contain one or two cis double bonds where the double bonds are disubstituted.
  • An alkynyl group contains at least one carbon-carbon triple bond and typically is C10-C30 in chain length.
  • the alkyl, alkenyl or alkynyl groups may be straight chain or branched.
  • other derivative groups containing alkyl moieties for example, alkoxy moieties and the like, also may contain short and/or long chain groups as appropriate in the context, unless otherwise defined.
  • L2 is C 4 -C 12 alkylene, optionally substituted at up to 2 positions by OR 9 .
  • X and Y independently may be selected from: L4-Het, where L4 is C1-C4 alkylene and Het is a C4-C12 heterocycle containing at least one nitrogen atom;
  • R 1 and R 2 independently may be H, C8-C20 alkyl, or monounsaturated C8-C20 alkenyl.
  • R R independently may be selected from H, C1 C6 alkyl, and C3 C6 cycloalkyl. In specific examples, R 3 -R 8 independently are H or C1-C3 alkyl.
  • R 9 may be H, -CO-C 8 -C 20 alkyl, -CO-monounsaturated C 8 -C 20 alkenyl, C 8 -C 20 alkyl or monounsaturated C8-C20 alkenyl. Specific examples include: where R 9 is C14-C18 alkyl or monounsaturated alkenyl; where R 9 is H and R 1 and R 2 are not H; where R 9 is not H and R 1 and R 2 are H; and where R 9 is C 14 -C 18 alkyl or –CO-C 14 -C 18 alkyl and R 1 and R 2 are H.
  • the double bond in R 9 when present, is a cis double bond.
  • L1 and L3 indepedently may be the same or different
  • R 1 and R 2 may be the same or different.
  • L 2 may be C 4 -C 12 alkylene.
  • L2 is -CH2CH(OR 9 )CH(OR 9 )CH2-.
  • the molecules may contain heterocyclic moieties linked via an amide or carbamate linkage, where L1 is –CO- or -CO2- and X is L4Het, where Het is a heterocyclic ring as defined below.
  • the molecule of structure Formula IV may be symmetrical or non-symmetrical with regard to each or all of the substituents R 1 , R 2, L 2-3 and X and Y independently; that is each R 1 may be the same or different, each R 2 may be the same or different, L 1 and L 3 may be the same or different R 3 , and X and Y may be the same or different.
  • the structure of L2 need not be symmetrical.
  • n ese moecu es eac or or examp e may be, but is not limited to, C14-18 alkyl or C14-18 alkenyl; and each R 9 independently may be, but is not limited to, H, –(CO)C14-C18 alkyl, or –(CO)C 14 -C 18 alkenyl.
  • lipids described in U.S. Patent No: 8,034,977 are contemplated for use in the present compositions and methods (the contents of which is incorporated herein by reference in its entirety).
  • the lipid compositions can include at least a first ionizable lipid and optionally at least a first neutral lipid, wherein said lipid composition is suitable for forming a complex with a nucleic acid under aqueous conditions, wherein said the ionizable lipids have the structure of Formula (V): (Formula V), or salts thereof, ialkylphophatidyl, or X is .
  • d Z independently may be selected from the group consisting of alkoxy, alkanoyloxy, alkylamine, alkyl urethane and alkyl guanidine.
  • R 1 and R 2 independently may be selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 1 - C6 alkylamine, alkylaminoalcohol, spermiyl, spermidyl and carboxyspermiyl.
  • R 3 is H or C1- C4 alkyl.
  • R 4 , R 5 , and R 6 independently are selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, and alkylaryl, provided that at least one of R 4 , R 5 , and R 6 is a long chain alkyl or alkenyl.
  • the alkyl, alkenyl, aryl, and alkylaryl groups may contain, for example, 6 to 30 carbon atoms, advantageously 10 to 18 carbon atoms, although the skilled artisan will recognize that the groups may contain fewer than 6 or more than 30 carbon atoms.
  • W may be short chain (C 1 -C 6 ) alkyl or alkylamino, and R 7 may be a negative charge or short chain (C 1 -C 6 ) alkyl.
  • Example of the compounds of structure Formula V is the set of compounds having the structure Formula VA: (Formula VA), and salts thereof, , alkenyl or alkanoyl; Z 3 and Z 4 are independently C 1 -C 6 alkyl; R 1 and R 2 independently are selected from the group consisting of hydrogen, C1-C6 alkyl, alkylamine, alkylaminoalcohol, spermiyl, spermidyl and carboxyspermiyl, R 3 is H or C 1 -C 4 alkyl, and .
  • kyaminoalcohol lipids of Formula VA include, without limitation, Compound 5-1: d Compou , wherein each R independently is C 10 -C 18 alkyl or alkenyl.
  • additional ionizable lipids contemplated for use in the present compositions and methods are provided below (compounds X-1 to X-16) and any others from Figs 1 and 2 of Han et al (2021); “An ionizable lipid toolbox for RNA delivery; vol.12, page 7233; incorporated by reference in its entirety.
  • the lipid may have a positive or partial positive charge at physiological pH.
  • the ionizable lipid molecules are shown here for convenience in their neutral (unprotonated) forms, these molecules will exist in a partially or fully protonated form in solutions of appropriate pH, and that the present invention encompasses the molecules in all their protonated, unprotonated, ionized and non-ionized forms without limitation, unless specifically indicated otherwise.
  • the lipid compositions include one or more neutral co-lipids, although the skilled artisan will recognize that other co-lipids may be used.
  • the neutral lipid may be, for example, selected from the group consisting of a sterol or sterol derivative, a phospholipid, or a combination thereof.
  • the neutral lipid can be present at about 5-60 mol% of the overall lipid formulation. In some embodiments, neutral lipid(s) are present from about 15-50 mol%, e.g., 25- 40 mol %.
  • the amount of the neutral lipid in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % of the overall lipid formulation.
  • the lipid composition includes a neutral lipid, and the neutral lipid includes cholesterol.
  • the neutral lipid includes sterol.
  • the neutral lipid includes dioleoylphosphatidylethanolamine (DOPE).
  • DOPE dioleoylphosphatidylethanolamine
  • the neutral lipid includes diphytanoylphosphatidylethanolamine (DPhPE). In some embodiments, the neutral lipid includes Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3- phosphoethanolamine). In some embodiments, the neutral lipid includes Lyso-PC (1-acyl-3- hydroxy-sn-glycero-3-phosphocholine). In some embodiments, the neutral lipid includes distearoylphosphatidylcholine (DSPC). In some embodiments, the neutral lipid includes dioleoylphosphatidylcholine (DOPC). In some embodiments, the neutral lipid includes dipalmitoylphosphatidylcholine (DPPC).
  • DPPC dipalmitoylphosphatidylcholine
  • the neutral lipid includes palmitoyloleoylphosphatidylcholine (POPC). In some embodiments, the neutral lipid includes palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). In some embodiments, the neutral lipid includes dipalmitoyl phosphatidyl ethanolamine (DPPE). In some embodiments, the neutral lipid includes dimyristoylphosphoethanolamine (DMPE). In some embodiments, the neutral lipid includes distearoyl-phosphatidylethanolamine (DSPE).
  • POPC palmitoyloleoylphosphatidylcholine
  • POPE palmitoyloleoyl-phosphatidylethanolamine
  • DOPE-mal dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-
  • the neutral lipid includes 16-O-monomethyl PE. In some embodiments, the neutral lipid includes 16-O-dimethyl PE. In some embodiments, the neutral lipid includes 18-1-trans PE. In some embodiments, the neutral lipid includes 1-stearoyl-2-oleoyl-phosphatidyethanol amine (SOPE). In some embodiments, the neutral lipid includes 1,2-dioleoyl-sn-glycero-3-phophoethanolamine (trans DOPE). In some embodiments, the neutral lipid includes any combinations thereof.
  • Exemplary phospholipids useful in the compositions disclosed herein include, but are not limited to, dioleoylphosphoethanolamine (DOPE), diphytanolphosphatidylethanolamine (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), dipalmitoyl phosphati
  • Phospholipids useful in the compositions provided herein can be present, for example at about 5 mol% to about 20 mol% of the lipid composition formulation.
  • phospholipids are present at a range from about 1 mol % to about 40 mol%, e.g., from 1 mol% to about 25 mol %.
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 0.5 mol %, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol%, 10 mol%, 12 mol%, 14 mol%, 16 mol %, 18 mol%, or 20 mol %, or any amount in between, of the overall lipid composition formulations.
  • the phospholipids useful in the composition can be present from about 5 mol% to about 15 mol%.
  • the phospholipids useful in the composition can be present from about 5 mol% to about 10 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 5 mol% to about 12 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 35 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 30 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 25 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 20 mol%.
  • the phospholipids useful in the composition can be present from about 1 mol% to about 15 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 10 mol%. In some embodiments, the phospholipids useful in the composition can be present from about 1 mol% to about 5 mol%. [0230] In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 0.5 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 1 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 2 mol %.
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 3 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 4 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 5 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 10 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 12 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 15 mol %.
  • the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 16 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 17 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 18 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 19 mol %. In some embodiments, the amount of the phospholipid in the lipid composition formulations disclosed herein is at least about 20 mol %.
  • lipid composition formulations include sterols, or lipids containing sterol moieties (“sterol derivatives”).
  • sterols are a subgroup of steroids consisting of steroid alcohols.
  • Exemplary sterols and lipids containing sterol moieties useful in the lipid composition formulations provided herein include, but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
  • the structural lipid is a sterol.
  • Some lipid composition formulations provided herein include a sterol or sterol derivative.
  • the sterols or sterol derivatives can be present at about 5-60 mol% of the overall lipid composition formulation.
  • the sterol or sterol derivatives are present from about 15-50 mol%, e.g., 25-40 mol %.
  • the amount of the sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % of the overall lipid formulation.
  • Some lipid composition formulations provided herein do not include a sterol or sterol derivative.
  • the lipid complex compositions can 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, polyamidoamine, poly(beta- amino esters), oligoalkylamines, positively charged dendrimers and fractured dendrimers, cationic ⁇ -cyclodextrin containing polymers (CD-polymers), DEAE-dextran and the like.
  • PEI polyethyleneimine
  • CD-polymers cationic ⁇ -cyclodextrin containing polymers
  • the lipid complex compositions may include linear PEIs, branched PEIs, and/or derivatives or modified forms thereof, such as cyclodextrin-PEI, stearic acid-PEI, aromatic-PEI, histidinyl-PEI, and PEI-PEG. Linear PEI, branched PEI, and derivatives thereof are commercially available at a variety of molecular weights.
  • the lipid complex compositions e.g., lipid nanoparticles, liposomes and lipoplexes
  • the lipid complex compositions can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.
  • exosome refers to the small membrane vesicles secreted by most cells that contain cell specific payloads of proteins, lipids and, genetic material and other biomolecules that are transported to other cells in different location of the tissue. Exosomes can be considered liposomal particles. Exosomes or lipid mixtures obtained therefrom, can be used in combination with other transfection agents or helper lipid mixtures. Exosomes are also referred to as microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes and oncosomes.
  • lipid compositions provided herein can also include a stabilizing agent, such as a stabilizing lipid.
  • Stabilizing lipids can be neutral lipids, or they can be charged.
  • Stabilizing lipids that can advantageously be used in the formulations provided herein include, but are not limited to, polyethylene glycol (PEG)-modified lipids.
  • PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG- modified 1,2-diacyloxypropan-3-amines.
  • PEGylated lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • stabilizing lipids useful in the compositions disclosed herein include, e.g., polyglycol lipids, yoxyethylene alkyl ethers, diblock polyoxyethylene ether co- polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers.
  • the stabilizing agent is present at about 0.1 - 5 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol %, 3 mol%, 3.5 mol %, 4 mol %, 4.5 mol%, 5 mol%, or any value in between, of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 5 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 4 mol% of the lipid composition.
  • the stabilizing agent is present at about 0.5 mol% to about 3 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 0.5 mol% to about 2 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 0.5 mol% to about 1 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 5 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 4 mol% of the lipid composition. In other examples, the stabilizing agent is present at about 1 mol% to about 3 mol% of the lipid composition.
  • the stabilizing agent is present at about 1 mol% to about 2 mol% of the lipid composition.
  • payload is complexed to the exterior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • nucleic acid is complexed to the exterior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • the compositions have from about 20% to about 50% of the nucleic acid complexed to the exterior of the lipid complex.
  • the compositions have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed to the exterior of the lipid complex. Exterior complexation of a nucleic acid can be measured by methods know in the art, such as in Blakney et al. (2019) Gene Therapy 26:363-372. [0239]
  • payload is complexed on the interior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • nucleic acid is complexed on the interior of the lipid complex (e.g., liposomes, lipid nanoparticles).
  • the compositions have an encapsulation efficiency from about 75% to about 95%, or from about 85% to about 90%. In some examples, the encapsulation efficiency is from about 75% to about 100%. In some examples, the encapsulation efficiency is from about 75% to about 95%. In some examples, the encapsulation efficiency is from about 75% to about 90%. In some examples, the encapsulation efficiency is from about 75% to about 85%. In some examples, the encapsulation efficiency is from about 75% to about 80%. In some examples, the encapsulation efficiency is from about 80% to about 95%. In some examples, the encapsulation efficiency is from about 80% to about 90%. In some examples, the encapsulation efficiency is from about 80% to about 85%.
  • the lipid composition includes at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, or 0.2.
  • the lipid composition comprises an N/P ratio of 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0.
  • the lipid composition includes at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises LLELLESL (SEQ ID NO: 6), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio of 0.01 to 5.0, or from 0.01 to 0.2, or from 0.05 to 0.5, or from 0.1 to 1.0, or from 0.5 to 2.0, or from 1.0 to 5.0.
  • the administered lipid composition includes at least one ionizable lipid having a charge (N), at least one peptide, wherein the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6), and a nucleic acid molecule comprising a charge (P), wherein the composition comprises an N/P ratio 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, or 0.2.
  • the lipid composition comprises an N/P ratio of 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0.
  • the lipid composition comprises an N/P ratio from 0.01 to 1.0, from 0.5 to 2.0, or from 1.0 to 8.0.
  • the administered lipid composition further comprises at least one neutral lipid.
  • 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.
  • a transfection enhancing agent such as a peptide comprising SEQ ID NO: 25 and/or SEQ ID NO: 26.
  • the immune cell includes T cell, B cell, dendritic cell (DC), T helper cell, cytotoxic T cells (CTL), natural killer cell (NK), macrophage including tissue- specific macrophage populations, or combinations thereof.
  • the immune cell includes DCs.
  • the immune cell includes a spleen immune cell.
  • the lipid composition targets an immune cell of the subject of the subject 1.2x or more compared to targeting of a non-immune cell of the subject.
  • the lipid composition targets an immune cell of the subject of the subject 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2.0x, 2.5x or more compared to targeting of a non- immune cell of the subject.
  • the level that is determined may an increased level.
  • the term “increased” with respect to level refers to any % increase above a control level.
  • the increased level may be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, at least or about a 95% increase, relative to a control level.
  • the level that is determined may a decreased level.
  • the term “decreased” with respect to level refers to any % decrease below a control level.
  • the decreased level may be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 25% decrease, at least or about a 30% decrease, at least or about a 35% decrease, at least or about a 40% decrease, at least or about a 45% decrease, at least or about a 50% decrease, at least or about a 55% decrease, at least or about a 60% decrease, at least or about a 65% decrease, at least or about a 70% decrease, at least or about a 75% decrease, at least or about a 80% decrease, at least or about a 85% decrease, at least or about a 90% decrease, at least or about a 95% decrease, relative to a control level.
  • mice received lipid formulations containing 5 ⁇ g of firefly luciferase mRNA via intramuscular (IM) or intravenous (IV) routes, respectively.
  • IM intramuscular
  • IV intravenous
  • mice were injected with d- Luciferin (150 mg kg ⁇ 1, intraperitoneal) and imaged using an IVIS Lumina III system (Perkin Elmer) to observe the signal of luciferase.
  • IVIS Lumina III system Perkin Elmer
  • the luminescence intensities in each region of interest (ROIs) of the isolated organs were quantified using the Living Image 3.0 software (PerkinElmer, Waltham, USA).
  • ROIs region of interest
  • the targeted delivery of the immune cell to the spleen included biodistribution (described above), which confirmed efficient delivery specifically to the spleen after intravenous administration of luciferase mRNA. Targeted delivery was also evaluated using a reporter mRNA and analysis of efficiency by flow cytometry.
  • methods for delivering a payload to a spleen cell in a subject include: (i) admixing 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 payload-containing lipid complex to a subject.
  • methods for delivering a nucleic acid to a spleen cell in a subject include: (i) admixing at least one ionizable lipid, at least one peptide, wherein the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6); and a payload, to form a lipid complex; and (ii) administering the lipid complex to a subject.
  • at least one neutral lipid is mixed with the at least one ionizable lipid and at least one peptide to form the lipid complex.
  • the payload is a nucleic acid, for example an RNA or a DNA molecule.
  • the payload comprises a peptide or polypeptide molecule.
  • methods for expressing a protein in spleen tissue in a subject include administering the lipid composition (lipid complex) to the subject.
  • methods for preparing a population of lipid formulations containing a nucleic acid payload molecule include (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 injecting comprises extrusion, in-line mixing, microfluidic mixing, evaporation, or vortexing; and (c) producing the population of lipid formulations complexed with a nucleic acid payload.
  • the peptide is in an ethanol solution when added.
  • the lipid solution of (b) further comprises at least one neutral lipid.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the peptide is selected from the group consisting of SEQ ID NOs: 6-24.
  • step (a) further comprises a second peptide which comprises at least one of SEQ ID NO: 25 and/or SEQ ID NO: 26.
  • methods for preparing a population of lipid formulations containing a nucleic acid molecule payload include (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 the population of lipid formulations complexed with a nucleic acid payload.
  • the peptide and lipids are in an ethanol solution when mixed with the aqueous solution containing the nucleic acid molecules.
  • the lipid phase of (a) further comprises at least one neutral lipid.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the peptide is selected from the group consisting of SEQ ID NOs: 6-24.
  • step (a) further comprises contacting the components with a second peptide which comprises at least one of SEQ ID NO: 25 and/or SEQ ID NO: 26.
  • the preparation methods produce a population of lipid nanoparticles with a payload molecule encapsulated therein. In other embodiments, the preparation methods produce a population of liposomes with the payload encapsulated therein. In some embodiments, the preparation methods produce a population of lipid nanoparticles with a payload molecule complexed to the exterior of the lipid nanoparticle. In other embodiments, the preparation methods produce a population of liposomes with the payload molecule complexed to the exterior of the liposome.
  • the payload is a nucleic acid, for example an RNA or a DNA molecule. In other embodiments, the payload comprises a peptide or polypeptide molecule.
  • kits comprising a lipid composition.
  • the kit comprises the at least one ionizable lipid, and at least one peptide, wherein the peptide comprises the sequence LLELLESL (SEQ ID NO: 1) and reagents.
  • the peptide comprises at least 80% sequence identity to GLFEALLELLESLWELLLEA (SEQ ID NO: 6).
  • the kit further comprises at least one neutral lipid.
  • a lipid composition in the kit is suitable for delivery (e.g., local injection) to a subject.
  • the present invention also provides packaging and kits comprising pharmaceutical compositions for use in the methods of the present invention.
  • the kit can comprise one or more containers selected from the group consisting of a bottle, a vial, an ampoule, a blister pack, and a syringe.
  • the kit can further include one or more of instructions for use in treating and/or preventing a disease, condition or disorder of the present invention, one or more syringes, one or more applicators, or a sterile solution suitable for reconstituting a pharmaceutical composition of the present invention.
  • the kit comprising a lipid composition provides that the ionizable lipid and neutral lipid are in a separate container from the peptide.
  • the kit comprising a lipid composition provides that the ionizable lipid and neutral lipid are in the same container as the peptide.
  • EXAMPLES [0259] The following examples illustrate certain specific embodiments of the invention and are not meant to limit the scope of the invention. Embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are merely intended to illustrate embodiments and are not to be construed to limit the scope herein. The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.
  • Example 1 Lipid-peptide complex formulations having at least one ionizable lipid and at least one peptide were screened and assessed by various in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, cellular uptake, and response to expression of the payload encoded protein. mRNAs used included those encoding firefly luciferase (fLuc), Cre recombinase, GFP, and influenza virus hemagglutinin (HA).
  • fLuc firefly luciferase
  • Cre recombinase GFP
  • HA influenza virus hemagglutinin
  • At least one ionizable lipid, a neutral lipid, and the peptide SEQ ID NO: 6 were complexed with an mRNA payload using standard complexation or lipid nanoparticle formation procedures.
  • the formulations examined varied, for example, in the concentration of peptide, the N/P ratios, and/or in type of ionizable lipid. Table 3.
  • Exemplary lipid complex formulations Formulation Ionizable lipid type Lipid concentration Peptide concentration LP17 Formula II 2 mg/ml 0.2 mg/ml LP18 Formula II 2 mg/ml 0.4 mg/ml Formulation Ionizable lipid type Lipid Peptide concentration concentration [0262] Ionizable lipid concentrations in the formulations of Table 3 ranged from 0.8 mg/l to 2.0 mg/l and all of the formulations in Tables 3 – 5 included one or two neutral lipids. The ionizable lipid type refers to the structure formulas provided herein. [0263] The formulations in the examples were formulated using reverse evaporation or microfluidization.
  • Lipids were weighed out and dissolved in chloroform, followed by evaporation on rotary evaporator. Lipid film was hydrated with water. In some situations, liposomal preparation was passed through microfluidizer. Lipid formulation was incubated with peptide solution overnight and stored at 4 o C until use. [0264] For complexation with nucleic acid, lipid formulation and nucleic acid were diluted in buffer, mechanically mixed by pipetting and/or vortexing, and incubated at room temperature for 10-20 minutes prior to delivery in rodent models or cells. [0265] Female BALB/c mice aged 6-10 weeks old purchased from The Jackson Laboratory and were acclimatized for 7 days before study.
  • Example 2 Firefly luciferase mRNA was complexed with each lipid-peptide formulation. Mice were injected with 5 ⁇ g fLuc mRNA-formulated lipid complexes using intravenous tail vein injection in a total volume 100 ml. At 4 h post injection, mice were anesthetized with isofluorane anaesthsia and imaged 10 min after intraperitoneal injection of 100 ⁇ L Rediject D- Luciferin (Perkin Elmer). Bioluminescence imaging was quantified in vivo and ex vivo using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.
  • IVIS Lumina III imaging system Perkin Elmer
  • Formulations LP4, LP5 and LP6 all contain the same ionizable lipid and differ only in the amount of peptide.
  • the presence of the peptide in the formulation significantly improved expression of the payload mRNA in spleen tissue compared to the lipid formulation without the peptide. For example, compare flux for LP4 (no peptide) to that for LP5 and LP6 in FIG.1B.
  • Formulations LP7, LP8, LP9 and LP10 contain the same ionizable lipid and differ only in the amount of peptide. Although the overall expression in the spleen was less with the formulations shown in FIG.1C compared to that with the formulations shown in FIGS.1A and 1B, the presence of the peptide significantly improved the expression of the luciferase mRNA in spleen tissue for the Formula VA-based lipid formulations. For example, compare flux for LP7 (no peptide) to that for the formulations in FIG.1C. [0269] In vivo delivery performance of formulations LP11-LP22 with N/P ratios ranging from 0.1 to 5 was assessed following intravenous administration.
  • the spleen flux results are shown in FIG.2.
  • the formulation results were compared to that obtained using mRNA- complexed formulation LP11 without SEQ ID NO:6 peptide (“LP11 - no peptide” in FIG.2).
  • LP11-LP22 with N/P ratios ranging from 0.1 to 5
  • in vivo delivery performance was assessed following intramuscular administration.
  • the luciferase signal was localized at injection region (muscle) and luciferase flux quantitation results are shown in FIG.3.
  • formulation LP11 without SEQ ID NO: 6 peptide (“LP11 - no peptide” in FIG.3) was used as a control.
  • N/P ratio was varied in fLuc mRNA-complexed LP11 and LP12 formulations and the effect on spleen delivery and biodistribution following intravenous injection was assessed. As shown in FIG.4A, N/P ratios as low as 0.125 with these formulations were effective in delivering fLuc mRNA expression in the spleen. These formulations also resulted in delivery specifically to the spleen, rather than to the liver or lung (FIG.4B).
  • Example 3 tdTomato reporter mice were used to analyze spleen cell populations targeted by the lipid-peptide formulations. A schematic for the workflow and analysis is shown in FIG.5. Cre mRNA was complexed with LP11 and LP12 formulations and the complexes administered to Ai14 mice intravenously. Five days after injection, the animals were killed and the spleens harvested. [0273] For splenocyte analysis, single-cell suspensions were prepared by passing the spleens through 70- ⁇ m cell strainers followed by lysis of blood cells with ACK Lysing Buffer and washing two times with PBS.
  • Splenocytes were partitioned into two staining groups and stained fluorescent antibodies for CD8 (APC), CD4 (PE), and B220 (FITC) or F480 (APC), CD11b (FITC), and CD11c (PE). Fluorescence of BDK-CART transfection in subpopulations was detected was detected on LSR-II.UV (BD Biosciences) using the Pacific Blue channel.
  • the Cre mRNA containing LP11 formulations delivered functional Cre mRNA to the dendritic cell population of the spleen. Delivery to dendritic cells of the spleen was significantly greater than delivery to the splenic B and T cell populations. A similar pattern was observed when GFP mRNA was used for delivery.
  • both LP11 and LP12 formulations were more effective in Cre mRNA delivery to all immune cell types of the spleen analyzed (as indicated by tdTomato expression) than either LP11 and LP12 formulations without SEQ ID NO: 6 peptide (FIG.7A). As also shown in FIG. 7A, both LP11 and LP12 formulations were significantly more effective in delivering the mRNA to dendritic cells as compared to the other immune cells of the spleen.
  • FIG.7B shows representative flow cytometric gating results in the dendritic cell population (MHCII/CD11c) versus tdTomato expression.
  • Firefly luciferase (fLuc) mRNA was formulated with LP11 formulation at varying N/P ratios.
  • fLuc mRNA was formulated with LP11 formulation without SEQ ID NO: 6 peptide at the same N/P ratios.
  • the mRNA containing compositions were administered via intravenous (IV) injection and imaged at 4 h post-injection as described in Example 2.
  • FIGs.8A and 8B show exemplary results of the LP11 formulation and LP11 without peptide formulation with N/P ratios from 0.25 to 4.0. At N/P ratios of 1.0 to 4.0, the LP11 formulations showed enhanced over that of LP11 without peptide formulations.
  • Example 4 Intracellular endosomal uptake and release of nucleic acid-containing lipid complexes were examined using workflows depicted in FIGs.9 and 11. For this analysis, HEK293 cells were transfected with Cy5-labeled mRNA complexed LP formulation or LP11 without SEQ ID NO: 6 peptide formulation and incubated at 37oC 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.
  • FIG.10A Exemplary results of uptake of mRNA-lipid complexes are shown in FIG.10A, with red fluorescence indicating Cy5-mRNA and blue fluorescence indicating DAPI. Quantitation of the cellular uptake over time is shown in FIG.10B.
  • the LP11 formulation internalized at an earlier time relative to the LP1 1 formulation without peptide, indicating a more efficient cellular uptake for the complete LP11 formulation.
  • HEK293 cells were transfected with Cy5-labeld mRNA complexed LP formulation or LP11 without SEQ ID NO: 6 peptide formulation and incubated at 37°C and 5% CO 2 for 3 hours. 50 nM LysoTrackerTM Green DND- 26 (Invitrogen) was added to the cells for 30 minutes before fixation to identify late endosome in lysosomal degradation (FIG. 11). The cells were then fixed and counterstained with DAPI as above, and images were acquired using a confocal laser-scanning microscope.
  • FIG. 12 Exemplary results of endosomal escape of mRNA-lipid complexes are shown in FIG. 12, with red fluorescence indicating Cy5-mRNA (left two panels), green fluorescence indicating late endosomal marker (center two panels) and blue fluorescence indicating DAPI (right two panels).
  • the LP11 formulation showed a more efficient endosomal escape than the LP11 formulation without peptide. As seen in the merged image (FIG. 12, right two panels), much less mRNA appeared free from LysoTracker staining using the complete LP11 formulation compared to the LP11 formulation without peptide.
  • Immunogenicity is defined as the ability of a substance (e.g., vaccine) to elicit an adaptive immune response.
  • Humoral immunity and cell-mediated immunity are two types of an adaptive immune response that enables a body to defend itself in a targeted way against foreign substance (antigen).
  • Humoral immune response is an antibody-mediated response that occurs when antigens are detected in the body. This mechanism is primarily driven by B cells that produce antibodies after the detection of a specific antigen. Unlike humoral immunity, cellular immunity does not depend on antibodies.
  • Cell-mediated immunity is primarily driven by mature T cells and the release of various cytokines (e g., IFN- ⁇ ) in response to an antigen.
  • Lipid-mRNA complexes of H10N8 influenza Hemagglutinin (HA) mRNA complexed LP11 or LP12 formulations were prepared.
  • the mRNA containing complexes were administered to BALB/c mice either intramuscularly (IM) or intravenously (IV) according to prime-boost immunization schedules with a 4-week interval.
  • FIG. 13 indicates the immunization schedule, the mRNA doses and routes of complex administration, and subsequent assays performed for assessing humoral and cellular immune responses.
  • the mRNA doses shown in FIG. 13 relate to those shown in FIGs.
  • 0.02 ⁇ g is 0.01 mg/kg (mRNA amount/mouse weight); 1 ⁇ g is 0.05 mg/kg, 5 ⁇ g is 0.25 mg/kg, 10 ⁇ g is 0.5 mg/kg, and 20 ⁇ g is 1 mg/kg.
  • HA-specific cellular responses by interferon-y IFN-y
  • splenocytes were isolated from the spleens at the end of prime-boost immunization schedule (week 6).
  • HA-specific peptide stimulation was used for T cell activation in in vitro culture and released IFN- ⁇ was detected by enzyme-linked immunosorbent spot (ELISPOT) assay.
  • ELISPOT enzyme-linked immunosorbent spot
  • FIGs. 15A-15C Exemplary results are shown in FIGs. 15A-15C.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Sont prévus des compositions, des procédés et des kits pour induire une réponse immunitaire chez un sujet. Selon certains aspects, une composition lipidique est décrite, qui comprend au moins un lipide ionisable comprenant une charge (N), au moins un peptide, et une molécule d'acide nucléique comprenant une charge (P). Selon certains aspects, des procédés sont prévus pour l'administration d'une charge utile à une cellule immunitaire en utilisant une composition lipidique comprenant au moins un lipide ionisable, au moins un peptide de libération endosomale, et une charge utile.
EP23750861.9A 2022-06-30 2023-06-29 Compositions lipidiques pour administration in vivo Pending EP4547282A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202263357584P 2022-06-30 2022-06-30
US202363510733P 2023-06-28 2023-06-28
PCT/US2023/069418 WO2024006937A1 (fr) 2022-06-30 2023-06-29 Compositions lipidiques pour administration in vivo

Publications (1)

Publication Number Publication Date
EP4547282A1 true EP4547282A1 (fr) 2025-05-07

Family

ID=87554381

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23750861.9A Pending EP4547282A1 (fr) 2022-06-30 2023-06-29 Compositions lipidiques pour administration in vivo

Country Status (4)

Country Link
EP (1) EP4547282A1 (fr)
JP (1) JP2025524562A (fr)
CN (1) CN119604312A (fr)
WO (1) WO2024006937A1 (fr)

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1129064B1 (fr) 1998-11-12 2008-01-09 Invitrogen Corporation Reactifs de transfection
JP4846588B2 (ja) 2003-05-22 2011-12-28 モレキュラー、トランスファー、インコーポレイテッド 核酸のトランスフェクションのための新規脂質
US7915230B2 (en) 2005-05-17 2011-03-29 Molecular Transfer, Inc. Reagents for transfection of eukaryotic cells
WO2012142622A1 (fr) 2011-04-15 2012-10-18 Molecular Transfer, Inc. Agents pour une administration améliorée d'acides nucléiques à des cellules eucaryotes
WO2013158127A1 (fr) * 2012-04-16 2013-10-24 Molecular Transfer, Inc. Agents permettant d'améliorer l'administration d'acides nucléiques dans des cellules eucaryotes
US9856496B2 (en) 2013-12-12 2018-01-02 Life Technologies Corporation Membrane-penetrating peptides to enhance transfection and compositions and methods for using same
LT3341027T (lt) * 2015-08-28 2024-06-25 Molecular Transfer, Inc. Transfekcijos kompleksai ir jų panaudojimo būdai
EP3423438A4 (fr) 2016-03-01 2020-05-06 Molecular Transfer, Inc. Protéines de mouvement de phytovirus et leurs procédés d'utilisation
EP3675826B1 (fr) 2017-08-31 2023-05-31 Life Technologies Corporation Compositions lipidiques cationiques pour administration spécifique à un tissu
CN119137109A (zh) * 2021-08-12 2024-12-13 生命技术公司 用于核酸递送的脂质

Also Published As

Publication number Publication date
WO2024006937A1 (fr) 2024-01-04
JP2025524562A (ja) 2025-07-30
CN119604312A (zh) 2025-03-11

Similar Documents

Publication Publication Date Title
US20250213718A1 (en) Lipids for nucleic acid delivery
US10953095B2 (en) Lipid compositions
US9139554B2 (en) Amino lipids and methods for the delivery of nucleic acids
ES2968649T3 (es) Nanopartículas lipídicas para la administración de ARNm en los pulmones
ES2911653T3 (es) Composiciones de nanopartículas lipídicas y métodos para el suministro de ARNm
AU2015210364B2 (en) Improved amino lipids and methods for the delivery of nucleic acids
US20110117125A1 (en) Compositions and methods for the delivery of nucleic acids
US20220370624A1 (en) Lipid compositions comprising peptide-lipid conjugates
Zhang et al. Hybrids of nonviral vectors for gene delivery
CN117136074A (zh) 适用于核酸递送的脂质
CN114073677A (zh) 一种脂质纳米颗粒
US20220378702A1 (en) Peptide-lipid conjugates
EP4565572A1 (fr) Lipides pour l'administration d'acides nucléiques
WO2025034764A1 (fr) Lipides pour l'administration d'acides nucléiques à des cellules eucaryotes
US20240000966A1 (en) Lipid Compositions For In Vivo Delivery
EP4547282A1 (fr) Compositions lipidiques pour administration in vivo
WO2025034802A2 (fr) Agents pour l'administration d'acides nucléiques
Ilies et al. Pyridinium amphiphiles in gene delivery–Present and perspectives
HK1160459B (en) Improved amino lipids and methods for the delivery of nucleic acids

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250106

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)