Lipid nanoparticle for targeted delivery

Abstract

Disclosed are compositions, methods, and kits related to lipid nanoparticles (LNPs) comprising ionizable lipids. The LNPs can comprise mRNA encoding therapeutic peptides for immunotherapy. The LNPs can preferentially deliver a therapeutic payload to a target organ.

Description

[0001] Lipid Nanoparticle for Targeted Delivery

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Nos: 63 / 564,352, filed March 12, 2024; and 63 / 626,978, filed January 30, 2024; the entire contents of each application is hereby incorporated by reference.

[0004] BACKGROUND

[0005] Synthetic mRNA provides a template for the synthesis of any given peptide, protein or protein fragment and lends itself to a broad range of pharmaceutical applications, including different modalities of cancer immunotherapy. To function in vivo, mRNA requires safe, effective, and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release. Lipid nanoparticles have successfully entered the clinic for the delivery of mRNA; in particular, lipid nanoparticle-mRNA vaccines have been approved in clinical use against coronavirus disease 2019 (COVID-19), which marks a milestone for mRNA therapeutics. Many other lipid nanoparticle-mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases.

[0006] SUMMARY

[0007] In an aspect, the present disclosure provides a composition, comprising a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid has an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein:

[0008] *indicates a point of attachment to a nitrogen in the amine headgroup;

[0009] Ri and R2 are each independently a C1-C12 bivalent aliphatic or hetero aliphatic radical;

[0010] X is , in which each of L3, L4, L5, and Lr, is, independently, a bond, O, S, or NRC; G is O, S, or NRd; Q is ORe, SRf, or NRgRh; and each of r and t is independently 1-6; each of Rc, Rd, Re, Rf, Rg, and Rhis independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl;

[0011] Y and U are each independently a bond, O, S, NR10, or Se; n is 0 or 1 ;

[0012] R3 and R4 are each independently H, Cl -CIO alkyl, Cl -CIO heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atom to which they are attached, form C=O;

[0013] R5is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for immunotherapy.

[0014] In some embodiments, the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra”’ are each independently, H, Cl -20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

[0015] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein: i) Rbis a substituted or unsubstituted alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; ii) nl and n2 are each independently 1, 2, 3, 4, 5, or 6; and iii) Rbl, Rb2, Rb3and Rb4are each independently H, or RLiPid wherein at least one of Rbl, Rb2, Rb3and Rb4is not H.

[0016] In some embodiments, the amine head group is selected from the group consisting of

[0017] In some embodiments, the hydrophobic tail RLipidhas a structure of wherein Rki and Rk3 are each independently a C1-C10 alkyl;

[0018] Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, Cl- C20 heterocycloalkyl, aryl, or heteroaryl;

[0019] Rk4 and Rks are each independently H, or C1-C10 alkyl.

[0020] In some embodiments, the lipid composition further comprises a steroid. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the lipid composition further comprises a helper lipid. In some embodiments, the helper lipid is

[0021] 1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC). In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG conjugated lipid. In some embodiments, the polymer conjugated lipid is 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000 (DSPE-PEG2k) or

[0022] 1.2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some embodiments, the lipid composition comprises a transactivator of transcription (TAT) peptide modification.

[0023] In some embodiments, the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%. In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.

[0024] In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16 / 4 / 1 / 1.

[0025] In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.8 / 4 / 2 / 1.

[0026] In some embodiments, the weight ratio of the pharmaceutical agcnt / lipid composition is from about 1:200 to about 1:5.

[0027] In some embodiments, the lipid composition further comprises a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid is about 2 / 1 / 1.

[0028] In some embodiments, the lipid composition further comprises an excipient. In some embodiments, the excipient is selected from the group consisting of (2-hydroxypropyl)-P- cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, 80-EC16, and Bile salts. In some embodiments, the excipient is present in the composition at a weight percentage from about 5% to about 60%.

[0029] In some embodiments, the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof. In some embodiments, the pharmaceutical agent comprises: (a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or (b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof.

[0030] In some embodiments, the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid.

[0031] In some embodiments, the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease.

[0032] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical.

[0033] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical.

[0034] In an aspect, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering an effective amount of any composition disclosed in this application.

[0035] In an aspect, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition disclosed herein that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to that achieved absent the lipid composition.

[0036] In an aspect, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according disclosed herein that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to a non-target organ.

[0037] In some embodiments, the administering is through systemic administration. In some embodiments, the administering is through intramuscular administration. In some embodiments, the administering is through intravenous administration. In some embodiments, the pharmaceutical agent is delivered at a dosage of no more than 3 mg / kg body weight. In some embodiments, the pharmaceutical agent is delivered at a dosage from about 1 pg / kg body weight to about 3 mg / kg body weight. In some embodiments, the pharmaceutical agent is delivered in one or more doses.

[0038] In some embodiments, the pharmaceutical agent is delivered for cancer immunotherapy .

[0039] In another aspect, the present disclosure provides a compound represented by Formula (III):

[0040] Head - (RLipid)P(Formula III) or a pharmaceutically acceptable salt thereof; wherein: wherein each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each RL1Pldis independently selected from: each Rki and Rk3 are independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4and Rks are independently selected from H and C1-C10 alkyl; and s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6.

[0041] In some embodiments, RLipidis selected from the group consisting of

[0042] In some embodiments, Head is selected from the group consisting of some embodiments, RL1Pldis represented by a structure in

[0043] TABLE 1 or TABLE 3. In some embodiments, the compound is represented by a structure in TABLE 2 or TABLE 4. In some embodiments, RL1Pldis represented by a structure in TABLE 3. In some embodiments, the compound is represented by a structure in TABLE 4.

[0044] In some aspects, the present disclosure provides a composition comprising a pharmaceutical agent assembled with a lipid composition comprising an ionizable lipid represented by Formula (III):

[0045] Head - (RLipid)P(Formula III) or a pharmaceutically acceptable salt thereof; wherein: wherein each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each RL1Pldis independently selected from: each of Rki and Rk3 are independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4and Rks are independently selected from H and C1-C10 alkyl; s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6. In some aspects, the present disclosure provides a composition, comprising a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid has an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine headgroup;

[0046] Ri and R2 are each independently selected from C1-C12 alkylene, C1-C12 alkenylene, and 1- to 12- membered heteroalkylene;

[0047] X is , in which each of L3, L4, L5, and Lr, is, independently, a bond, O, S, or NRC; G is O, S, or NRd; Q is ORe, SRf, or NRgRh; r is selected from 1, 2, 3, 4, 5, and 6; and t is t is selected from 1, 2, 3, 4, 5, and 6; each of Rc, Rd, Re, Rf, Rg, and Rhis independently H, Cl -CIO alkyl, 1- to 10- membered heteroalkyl, aryl, or heteroaryl;

[0048] Y and U are each independently a bond, O, S, NR10, or Se; n is 0 or 1 ;

[0049] R3 and R4 are each independently H, C1-C10 alkyl, 1- to 10-membered heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atom to which they are attached, form =0;

[0050] R5is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20- membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for immunotherapy.

[0051] In some embodiments, the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C6-C20 aryl, and 6- to 20-membered heteroaryl, or a RL1Pld selected from C1-C20 alkylene, a 1- to

[0052] 20-membered heteroalkylene, arylene, and heteroarylene.

[0053] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein: i) Rbis a substituted or unsubstituted Ci-Ce alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; and ii) nl and n2 are each independently 1, 2, 3, 4, 5, or 6.

[0054] In some embodiments, the amine head group is selected from the group consisting of

[0055] In some embodiments, the hydrophobic tail RL1Pldhas a structure of

[0056] wherein each Rki and Rk3 is independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl,

[0057] C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; and each Rk4and Rks is independently selected from H and C1-C10 alkyl.

[0058] In some embodiments, the lipid composition further comprises a steroid. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the lipid composition further comprises a helper lipid. In some embodiments, the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC). In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG conjugated lipid. In some embodiments, the polymer conjugated lipid is 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some embodiments, the lipid composition comprises a transactivator of transcription (TAT) peptide modification. In some embodiments, the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%. In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 20%. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16 / 4 / 1 / 1. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.8 / 4 / 2 / 1. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.6 / 4 / 2 / 1. In some embodiments, the weight ratio of the pharmaceutical agcnt / lipid composition is from about 1:200 to about 1:5. In some embodiments, the lipid composition further comprises a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid is about 2 / 1 / 1.

[0059] In some embodiments, the lipid composition further comprises an excipient. In some embodiments, the excipient is selected from the group consisting of (2-hydroxypropyl)-P- cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, 80-EC16, and Bile salts. In some embodiments, the excipient is present in the composition at a weight percentage from about 5% to about 60%. In some embodiments, the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof. In some embodiments, the pharmaceutical agent comprises: (a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or (b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof. In some embodiments, the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid. In some embodiments, the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical.

[0060] In some aspects, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering an effective amount of any composition disclosed in this application.

[0061] In some aspects, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition disclosed herein that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to that achieved absent the lipid composition.

[0062] In some aspects, the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according disclosed herein that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to a non-target organ.

[0063] In some embodiments, the administering is through systemic administration. In some embodiments, the administering is through intramuscular administration. In some embodiments, the administering is through intravenous administration. In some embodiments, the pharmaceutical agent is delivered at a dosage of no more than 3 mg / kg body weight. In some embodiments, the pharmaceutical agent is delivered at a dosage from about 1 pg / kg body weight to about 3 mg / kg body weight. In some embodiments, the pharmaceutical agent is delivered in one or more doses. In some embodiments, the pharmaceutical agent is delivered for cancer immunotherapy. In some embodiments, the target organ comprises a liver. In some embodiments, the target organ comprises a spleen or a lymph node. INCORPORATION BY REFERENCE

[0064] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0065] BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0067] FIGs. 1A-1B illustrate synthesis schemes of lipid tails. FIG. 1A depicts the synthesis of lipid tail O12B. FIG. IB depicts the synthesis of lipid tail Compound 4 comprising a carbonate.

[0068] FIGs. 2A-2G show whole body bioluminescence from IVIS imaging of adult BALB / c mice at about 6 hours post single i.v. injections of luciferase mRNA / LNPs.

[0069] FIGs. 3A-3C depict plasma concentrations of hEPO in CD1 mice at 6 hours post single IM injections of hEPO mRNA / LNPs.

[0070] FIGs. 4A-4B show whole body bioluminescence from IVIS imaging of adult BALB / c mice at about 6 hours post single i.v. injections of luciferase mRNA / LNPs. Lipid nanoparticles were formulated with luciferase mRNA and lipids L88, L854, and L855, respectively. IVIS images and structures of the three lipids are shown in FIG. 4A. Quantification of luciferase intensity is shown in FIG. 4B. The experiment was conducted in duplicates. However, due to lab error the second mouse intended for testing the luciferase mRNA / L854-LNP did not receive the full injection.

[0071] FIGs. 5A-5B show whole body bioluminescence from IVIS imaging of adult BALB / c mice at about 6 hours post single i.v. injections of luciferase mRNA / LNPs. Lipid nanoparticles were formulated with luciferase mRNA and lipids L88, L854, and L855, respectively. IVIS images are shown in FIG. 5A. Quantification of luciferase intensity is shown in FIG. 5B. The experiment was conducted in duplicates.

[0072] FIGs. 6A-6B show whole body bioluminescence from IVIS imaging of adult BALB / c mice at about 6 hours post single i.v. injections of luciferase mRNA / LNPs. Lipid nanoparticles were formulated with luciferase mRNA and lipids L854 and ALC-0315, respectively. IVIS images are shown in FIG. 6A. Quantification of luciferase intensity is shown in FIG. 6B. The experiment was conducted in duplicates.

[0073] FIGs. 7A-7B show whole body bioluminescence from IVIS imaging of adult BALB / c mice at about 6 hours post single i.v. injections of luciferase mRNA / LNPs. Lipid nanoparticles were formulated with luciferase mRNA and lipids L855 and SM-102, respectively. IVIS images are shown in FIG. 7A. Quantification of luciferase intensity is shown in FIG. 7B. The experiment was conducted in duplicates.

[0074] FIGs. 8A-8B show ex vivo IVIS imaging of the spleen at about 5 hours post single i.v. injections of luciferase mRNA / LNPs.

[0075] FIG. 9 depicts cell type specificity of the spleen-targeting LNPs. More TdTomato (+) cells were seen in antigen presenting cells (APCs) located in the spleen, indicating that the Luc mRNA / LNPs delivered mRNA more efficiently to APCs.

[0076] FIG. 10 show ex vivo IVIS imaging of the liver at about 5 hours post single i.v. injections of luciferase mRNA / LNPs.

[0077] DETAILED DESCRIPTION

[0078] The present disclosure provides compositions, methods, and kits related to lipid nanoparticles (LNPs) comprising ionizable lipids and mRNA encoding therapeutic peptides. In some embodiments, the LNPs comprise mRNA that encode therapeutic peptides (e.g., antigen-binding peptide, antigen receptors, or immunomodulators) for immunotherapy.

[0079] The present disclosure also provides ionizable lipids and compositions, methods, and kits related to lipid nanoparticles (LNPs) formulated by the ionizable lipids and a therapeutic payload. In some embodiments, the therapeutic payload comprises a therapeutic peptide or a nucleic acid molecule encoding the therapeutic peptide. In some embodiments, the LNPs comprise an mRNA that encodes a therapeutic peptide for immunotherapy. In some embodiments, the therapeutic peptide for immunotherapy comprises an antigen-binding peptide, an antigen receptor, or an immunomodulators. In some embodiments, the LNPs can deliver the therapeutic payload to a target organ or cell. In some embodiments, the target organ comprises a liver or a spleen.

[0080] Definitions

[0081] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure.

[0082] In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise:

[0083] As used throughout the specification and claims, the terms “a”, “an” and “the” are generally used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. For example, a “cleavage sequence”, as used herein, means “at least a first cleavage sequence” but includes a plurality of cleavage sequences. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present application.

[0084] The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0085] As used herein, the term “antibody” refers to an immunoglobulin (Ig) whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen-binding domain. The term further includes “antigen-binding fragments” or “functional fragment thereof’, or “fragment of an antibody”, “antibody fragment”, “functional fragment of an antibody” and other interchangeable terms for similar binding fragments such as described below. An antibody includes, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity-matured antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), heteroconjugate antibodies, antibody fragments, and combinations thereof (e.g., a monoclonal antibody that is also deimmunized, a humanized antibody that is also deimmunized, etc.). An antibody can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen binding fragment can include, for example, Fab’, F(ab’)2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, or nanobody.

[0086] The term “antigen” refers to a molecule bound by an antibody or a fragment thereof. Antigens may be proteins recognized by immunoglobulins, in which case the sites on the proteins bound by the immunoglobulins are referred to as “epitopes”. Antigens may also, or alternatively, be recognized by non-immunoglobulin ligand binding domain of an antigenbinding peptide. In such cases, "antigen" refers to binding partners of the non-immunoglobulin ligand binding domain of the bispecific binding peptide. In some cases, the antigen can be a ligand of a cell surface receptor, e.g., a ligand of NKG2D. In some cases, the bispecific antigenbinding peptide contains a cell surface receptor ligand, or receptor binding portion thereof, and the antigen is the ligand binding portion of the cell surface receptor.

[0087] As used herein, the terms “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms generally refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms or improvement in one or more clinical parameters associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0088] A “therapeutic effect” or “therapeutic benefit,” as used herein, generally refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the disclosure other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0089] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, generally refer to an amount of a drug or a biologically active protein, either alone or as a part of a polypeptide composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0090] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” defines the exact number (n) of carbon atoms in the group / class. “C<n” defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number as small as possible for the group / class in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(C<8)” or the class “alkene(C<8)” is two. Compare with “alkoxy(C<10)”, which designates alkoxy groups having from 1 to 10 carbon atoms. “Cm-n” or “Cm-Cn” defines both the minimum (m) and maximum number (n) of carbon atoms in the group. Thus, “C1-C10 alkyl” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C5 olefin”, “C5- olefin”, “olefin^)”, and “olefines” are all synonymous.

[0091] The term “saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of ketoenol tautomerism or imine / enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0092] The term “aliphatic” generally signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In aliphatic compounds / groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, that is joined by single carbon-carbon bonds (alkanes / alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes / alkenyl) or with one or more carbon-carbon triple bonds (alkynes / alkynyl).

[0093] The term “aromatic” when used to modify a compound or a chemical group atom means the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.

[0094] The term “alkyl” when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH3)2 (z-Pr, ‘Pr or isopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), - C(CH3)3 (tert-butyl, t-butyl, t-Bu or 'Bu), and -CH2C(CH3)3 (zzeo-pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(0)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2C1, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2CI. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (z.e. -F, -Cl, -Br, or -I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH2CI is a nonlimiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are nonlimiting examples of fluoroalkyl groups.

[0095] The term “cycloalkyl” when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2(cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term “cycloalkanediyl” when used without the “substituted” modifier refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group is a non_ limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2,

[0096] -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0097] The term “alkenyl” when used without the “substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carboncarbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2(vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2(allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term “alkenediyl” when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “a-olefin” are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHC1 and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0098] The term “alkynyl” when used without the “substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C=CH, -C=CCH3, and -CH2C=CCH3 are non-limiting examples of alkynyl groups. An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl. When any of these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0099] The term “aryl” when used without the “substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, the carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl. The term “arenediyl” when used without the “substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). Non-limiting examples of arenediyl groups include:

[0100] The term “aralkyl” when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and / or the aryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Nonlimiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl- eth-l-yl.

[0101] The term “hetero” when used to modify a compound or chemical group means the compound or chemical group has at least an atom that is not carbon, for example, N, O, S, Se, P, Si, B, or any other heteroatom. For example, a heteroaliphatic can be any aliphatic moiety containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. A heterocycle can be any ring containing a ring atom that is not carbon. A heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms. A heterocycle can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.

[0102] The term “heteroaryl” when used without the “substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl rings may contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen, and sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term ' W-hctcroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. The term “heteroarenediyl” when used without the “substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system. Nonlimiting examples of heteroarenediyl groups include:

[0103] The term “heterocycloalkyl” when used without the “substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term “A-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. / V- yrrolidinyl is an example of such a group. The term “heterocycloalkanediyl” when used without the “substituted” modifier refers to a divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include:

[0104] When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0105] The term “acyl” when used without the “substituted” modifier refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as those terms are defined above. The groups, -CHO, -C(0)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6HS, -C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term “aldehyde” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the “substituted” modifier one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups, -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(0)NH2 (carbamoyl), and -CON(CH3)2, are non-limiting examples of substituted acyl groups.

[0106] The term “alkoxy” when used without the “substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3(tert-butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term “alkoxydiyl” refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The term “alkylthio” and “acylthio” when used without the “substituted” modifier refers to the group -SR, in which R is an alkyl and acyl, respectively. The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms is used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0107] The term “alkylamino” when used without the “substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3.

[0108] The term “dialkylamino” when used without the “substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl. Non-limiting examples of dialkylamino groups include: -N(CH3)2 and -N(CH3)(CH2CH3). The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”, “heterocycloalkylamino”, “alkoxyamino”, and “alkylsulfonylamino” when used without the “substituted” modifier, refers to groups, defined as -NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHCeHs. The term “alkylaminodiyl” refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(0)CH3. The term “alkylimino” when used without the “substituted” modifier refers to the divalent group =NR, in which R is an alkyl, as that term is defined above. When any of these terms is used with the “substituted” modifier one or more hydrogen atom attached to a carbon atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -NHC(0)0CH3 and -NHC(0)NHCH3 are non-limiting examples of substituted amido groups.

[0109] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Unless specified otherwise, aliphatic, heteroaliphatic, oxyaliphatic, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, hydroxyalkyl, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties.

[0110] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. Generally, the term “about,” as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass in one example variations of ± 20% or ± 10%, in another example ± 5%, in another example ± 3%, in another example ± 1%, and in yet another example ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0111] As used in this application, the term “average molecular weight” refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have different levels of polymerization and thus a different molar mass. The average molecular weight can be used to represent the molecular weight of a plurality of polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three major types of average molecular weight: number average molar mass, weight (mass) average molar mass, and Z- average molar mass. In the context of this application, unless otherwise specified, the average molecular weight represents either the number average molar mass or weight average molar mass of the formula. In some embodiments, the average molecular weight is the number average molar mass. In some embodiments, the average molecular weight may be used to describe a PEG component present in a lipid.

[0112] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0113] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

[0114] As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

[0115] The term “assemble” or “assembled,” as used herein, in context of delivery of a payload to target cell(s) generally refers to covalent or non-covalent interaction(s) or association(s), for example, such that a therapeutic or prophylactic agent be complexed with or encapsulated in a lipid composition.

[0116] As used herein, the term “lipid composition” generally refers to a composition comprising lipid compound(s), including but not limited to, a lipoplex, a liposome, a lipid particle. Examples of lipid compositions include suspensions, emulsions, and vesicular compositions.

[0117] As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0118] “Pharmaceutically acceptable salts” means salts of compounds of the present application which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphor sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl- substituted alkanoic acids, propionic acid, -tolucncsul Ionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0119] The term “pharmaceutically acceptable carrier,” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0120] “Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

[0121] The term “helper lipid” as used in this disclosure refers to a lipid that contributes to the stability or delivery efficacy of a lipid composition. A helper lipid can be a zwitterionic lipid, such as a phospholipid. A helper lipid can be phosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some cases, the term “helper lipid” refers to phospholipids or other zwitterionic lipids in the LNP composition. In some cases, when describing the formulation of an LNP using weight ratios of the lipid components (e.g., lipidoid, steroid, helper lipid, and polymer conjugated lipid), a helper lipid refers to a phospholipid or another zwitterionic lipid. For example, the weight ratio of the lipidoid / steroid / helper lipid / polymer conjugated lipid is about 14 / 4 / 1 / 1. “Helper lipid” can refer to any class of lipid molecules that improves the particle stability and fluidity of lipid nanoparticles (LNP). Several classes of molecules can be used as helper lipids such as phospholipids (e.g., phosphoethanolamine, phosphocholine), zwitterionic lipids, steroid derivatives, and polymer conjugated lipids (e.g., PEGylated lipid). Representative helper lipids include cholesterol, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), Phosphatidylcholine (PC), Methoxy- Polyethyleneglycol (MW 2k)-distearoylphosphatidylethanolamine (mPEG2k-DSPE), and 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG2k).

[0122] Compositions

[0123] In one aspect, provided herein is a composition comprising pharmaceutical agent (or therapeutic agent) assembled with a lipid composition. In some embodiments, the lipid composition comprises an ionizable lipid.

[0124] In some embodiments, the ionizable lipid comprises an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine head group;

[0125] Ri and R2 are each independently a C1-C12 bivalent aliphatic or hetero aliphatic radical;

[0126] X is , in which each of L3, L4, L5, and Lr, is, independently, a bond, O, S, or NRC; G is O, S, or NRd; Q is ORe, SRf, or NRgRh; and each of r and t is independently 1-6; each of Rc, Rd, Re, Rf, Rg, and Rhis independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl;

[0127] Y and U are each independently a bond, O, S, NR10, or Se; n is 0 or 1 ;

[0128] R3 and R4, are each independently H, Cl -CIO alkyl, Cl -CIO heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atom to which they are attached, form C=O;

[0129] R5is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for immunotherapy.

[0130] In some embodiments, n is 0. In some embodiments, n is 1.

[0131] In some embodiments, n is 1 and R3 and R4 together with the atom to which they are attached, form C=O. In some embodiments, Y is CH2 and U is O. In some embodiments, Y and U are both O. In some embodiments, U is CH2 and Y is O. In some embodiments, Y and U are NR10. In some embodiments, Y is O and U is NR10. In some embodiments, Y is S and U is NR10.

[0132] In some embodiments, n is 0, and Y and U are both S.

[0133] In some embodiments, n is 0, and one of Y and U is O.

[0134] In some embodiments, n is 0, and one of Y and U is Se.

[0135] In some embodiments, Ri is a C1-C12 alkyl, linear or branched. In some embodiments, Ri is a C1-C10 alkyl, linear or branched. In some embodiments, Ri is a C1-C8 alkyl, linear or branched. In some embodiments, Ri is a C1-C6 alkyl, linear or branched. In some embodiments, Ri is a C1-C4 alkyl, linear or branched. In some embodiments, Ri is a C2 alkyl, , yl. In some embodiments,

[0136] Ri is a C1-C12 heteroaliphatic radical.

[0137] In some embodiments, R2 is a C1-C12 alkyl, linear or branched. In some embodiments, R2 is a Cl -CIO alkyl, linear or branched. In some embodiments, R2 is a C1-C8 alkyl, linear or branched. In some embodiments, R2 is a C1-C6 alkyl, linear or branched. In some embodiments, R2 is a C1-C4 alkyl, linear or branched. In some embodiments, R2 is a C2 alkyl, , yl. In some embodiments,

[0138] R2 is a C1-C12 heteroaliphatic radical.

[0139] In some embodiments, the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, Cl -20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

[0140] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein: iv) Rbis a substituted or unsubstituted alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; v) nl and n2 are each independently 1, 2, 3, 4, 5, or 6; and vi) Rbl, Rb2, Rb3and Rb4are each independently H, or RLipid wherein at least one of Rbl, Rb2, Rb3and Rb4is not H.

[0141] In some embodiments, Rbis a C1-C6 alkyl, linear or branched. In some embodiments, Rbis a substituted C1-C6 alkyl, linear or branched. In some embodiments, a substituent comprises hydroxyl, carbonyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, cyclic amine, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In some embodiments, the substituent comprises hydroxyl, any suitable substituent thereof.

[0142] In some embodiments, nl is 1, 2, 3, 4, 5, or 6. In some embodiments, nl is 2 or 3.

[0143] In some embodiments, n2 is 1, 2, 3, 4, 5, or 6. In some embodiments, n2 is 2 or 3.

[0144] In some embodiments, nl and n2 are identical. In some embodiments, nl and n2 are different.

[0145] In some embodiments, both nl and n2 are 2. In some embodiments, both nl and n2 are

[0146] 3. In some embodiments, both nl and n2 are 4.

[0147] In some embodiments, Rblis not H. In some embodiments, Rb2is not H. In some embodiments, Rb3is not H. In some embodiments, Rb4is not H.

[0148] In some embodiments, at least two of Rbl, Rb2, Rb3and Rb4are not H. In some embodiments, at least three of Rbl, Rb2, Rb3and Rb4are not H. In some embodiments, none of Rbl, Rb2, Rb3and Rb4is H.

[0149] In some embodiments, the amine head group is selected from the group consisting of 9 wherein Rki and Rk3 are each independently a C1-C10 alkyl;

[0150] Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, Cl- C20 heterocycloalkyl, aryl, or heteroaryl;

[0151] Rk4 and Rks are each independently H, or C1-C10 alkyl.

[0152] In some embodiments, Rki is a C1-C10 alkyl, linear or branched. In some embodiments,

[0153] Rki is a C1-C4 alkyl, linear or branched. In some embodiments, Rki is a C2 alkyl, e.g.,

[0154] In some embodiments, Rki is a C3 alkyl, e.g., . In some embodiments, Rki is a C4 alkyl.

[0155] In some embodiments, Rk3 is a C1-C10 alkyl, linear or branched. In some embodiments,

[0156] Rk3 is a C1-C4 alkyl, linear or branched. In some embodiments, Rk3 is a C2 alkyl, e.g.,

[0157] In some embodiments, Rk3 is a C3 alkyl, e.g., . In some embodiments, Rk3 is a C4 alkyl.

[0158] In some embodiments, Rk4 is H. In some embodiments, Rk4 is C1-C10 alkyl. In some embodiments, Rk4 is C1-C4 alkyl. In some embodiments, Rk4 is C4-C10 alkyl.

[0159] In some embodiments, Rks is H. In some embodiments, Rks is C1-C10 alkyl. In some embodiments, Rks is C1-C4 alkyl. In some embodiments, Rks is C4-C10 alkyl.

[0160] In some embodiments, Rk2 is a C1-C20 alkyl. In some embodiments, Rk2 is a C2-C20 alkenyl. In some embodiments, Rk2 is a C2-C20 alkynyl. In some embodiments, Rk2 is a C3- C20 cycloalkyl. In some embodiments, Rk2 is a C1-C20 heteroalkyl. In some embodiments, Rk2 is a Cl- C20 heterocycloalkyl, aryl, or heteroaryl.

[0161] In some embodiments, the at least one hydrophobic tail is selected from the TABLE 1.

[0162] TABLE 1. Exemplary Hydrophobic Tail

[0163] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails. In some embodiments, the at least two hydrophobic tails are independently of structure . In some embodiments, the at least two hydrophobic tails are identical. In some embodiments, the at least two hydrophobic tails are not identical. In some embodiments, one of the at least two hydrophobic tails is different from the rest.

[0164] In some embodiments, the lipid composition comprises at least three hydrophobic tails. In some embodiments, the at least three hydrophobic tails are independently of structure . In some embodiments, the at least three hydrophobic tails are identical. In some embodiments, the at least three hydrophobic tails are not identical. In some embodiments, one of the at least three hydrophobic tails is different from the rest.

[0165] In some embodiments, the lipid composition comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are independently of structure . in some embodiments, the two hydrophobic tails are identical. In some embodiments, the two hydrophobic tails are not identical.

[0166] In some embodiments, the lipid composition comprises three hydrophobic tails. In some embodiments, the three hydrophobic tails are independently of structure . In some embodiments, the three hydrophobic tails are identical. In some embodiments, two of the three hydrophobic tails are identical and the third hydrophobic tail is different. In some embodiments, all three hydrophobic tails are different.

[0167] In some embodiments, the lipid composition comprises four hydrophobic tails. In some embodiments, the four hydrophobic tails are independently of structure insome embodiments, the four hydrophobic tails are identical. In some embodiments, three of the four hydrophobic tails are identical and the fourth hydrophobic tail is different. In some embodiments, two of the four hydrophobic tails are identical, the other two hydrophobic tails are identical, and the two are different from other two. In some embodiments, two of the four hydrophobic tails are identical while the other two are different from each other and are different from the two. In some embodiments, all four hydrophobic tails are different.

[0168] In some embodiments, the ionizable lipid is selected from TABLE 2.

[0169] TABLE 2. Exemplary Ionizable Lipid

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] In some embodiments, the composition provided herein further comprises a steroid. In some embodiments, the steroid comprises a cholesterol or a cholesterol derivative. In some embodiments, the composition provided herein further comprises a helper lipid. In some embodiments, the helper lipid comprises l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the composition provided herein further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some embodiments, the lipid composition further comprises a modification comprising a cellpenetrating peptide. In some cases, the cell-penetrating peptide is transactivator of transcription (TAT).

[0184] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid, a helper lipid, and a polymer conjugated lipid.

[0185] In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 80%, about 10% to about 90%, from about 20% to about 30%, from about 20% to about 30%, from about 20% to about 40%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 30% to about 40%, from about 30% to about 50%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about 80%, from about 30% to about 90%, from about 40% to about 50%, from about 40% to about 60%, from about 40% to about 70%, from about 40% to about 80%, from about 40% to about 90%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, from about 70% to about 80%, from about 70% to about 90%, or from about 80% to about 90%.

[0186] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0187] In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.

[0188] In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0189] In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 14 / 4 / 1 / 1, about 15 / 4 / 1 / 1, about 16 / 4 / 1 / 1, about 17 / 4 / 1 / 1, about 18 / 4 / 1 / 1, about 19 / 4 / 1 / 1, about 20 / 4 / 1 / 1, about 14 / 4 / 2 / 1, about 15 / 4 / 2 / 1, about 16 / 4 / 2 / 1, about 16.8 / 4 / 2 / 1, about 17 / 4 / 2 / 1, about 18 / 4 / 2 / 1, about 19 / 4 / 2 / 1, or about 20 / 4 / 2 / 1.

[0190] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid is about 1 / 1 / 1, 2 / 1 / 1, about 3 / 1 / 1, about 4 / 1 / 1, about 5 / 1 / 1, about 6 / 1 / 1, about 2 / 2 / 1, about 3 / 2 / 1, about 4 / 2 / 1, about 5 / 2 / 1, or about 6 / 2 / 1.

[0191] In some embodiments, the lipid composition further comprises an excipient. The excipient can comprise EC- 16, (2-hydroxypropyl)-P-cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, or Bile salts. In some embodiments, the excipient is present in the composition at a weight percentage from about 5% to about 60%. In some embodiments, the excipient is present in the composition at a weight percentage from about 1% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.

[0192] In some embodiments, the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof. In some embodiments, the polynucleotide is a messenger ribonucleic acid (mRNA).

[0193] In some embodiments, the pharmaceutical agent comprises a polynucleotide that encodes or is configured to regulate a target gene or a gene product thereof.

[0194] In some embodiments, the pharmaceutical agent comprises (a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or (b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof.

[0195] In some embodiments, the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid.

[0196] In some embodiments, the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease. In some embodiments, the heterologous endonuclease comprises a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease. In some embodiments, the polynucleotide comprising a sequence that encodes the heterologous endonuclease.

[0197] In some embodiments, the pharmaceutical agent (or therapeutic agent) comprises mRNA encoding at least one therapeutic peptide for immunotherapy.

[0198] In some embodiments, the pharmaceutical agent (or therapeutic agent) is delivered for cancer immunotherapy.

[0199] In some embodiments, pharmaceutical agent is assembled in the lipid composition at a weight ratio of the pharmaceutical agcnt / l ipid composition of from about 1 :200 to about 1 : 100, from about 1:200 to about 1:50, from about 1:200 to about 1:40, from about 1:200 to about 1:30, from about 1:200 to about 1:20, from about 1:200 to about 1:10, from about 1:200 to about 1:5, from about 1:200 to about 1:1, from about 1:100 to about 1:50, from about 1:100 to about 1:40, from about 1:100 to about 1:25, from about 1:100 to about 1:20, from about 1:100 to about 1:15, from about 1:100 to about 1:10, from about 1:100 to about 1:5 or from about 1:100 to about 1:1.

[0200] In some embodiments, the target gene or the gene product thereof is specific to or primarily found in a target organ (e.g., liver, spleen, or lymph nodes) or a target cell (e.g., a immune cell provided herein; e.g., a T cell) of a subject.

[0201] In some embodiments, the target gene or the gene product thereof is associated with a disease or disorder of the target organ or the target cell.

[0202] In some embodiments, the gene modulating moiety is configured to provide a modified expression profile of the target gene or the gene product thereof in a target organ (e.g., liver, spleen, lymph nodes) or a target cell (e.g., a immune cell provided herein; e.g., a T cell) of a subject.

[0203] In some embodiments, the composition is formulated for systemic or local administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for intramuscular administration.

[0204] Further Compositions

[0205] In some aspects, provided herein is a compound represented by Formula (III):

[0206] Head - (RLipid)P(Formula III) or a pharmaceutically acceptable salt thereof; wherein: wherein each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each Rki and Rk3 are independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4and Rk5 are independently selected from H and C1-C10 alkyl; and s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6.

[0207] In some embodiments of a compound or salt of Formula (III), Head is selected from alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, 1- to 10- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle. In some embodiments, each Rais independently selected from H, C1-C5 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, 1- to 10-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle. In some embodiments, each Rais independently selected from H, C1-C5 alkyl, C3-C10 cycloalkyl, 3- to 10-membered heterocycloalkyl, 1- to 10-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle.

[0208] In some embodiments of a compound or salt of Formula (III), Head is selected from

[0209]

[0210] In some embodiments of a compound or salt of Formula (III), each Rais independently selected from H, methyl,

[0211] , and methyl, . In some embodiments, each Rais

[0212] In some embodiments of a compound or salt of Formula (III), Head is selected from ?wherein each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20- membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle. In some embodiments, Head is selected from selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20- membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7- membered heterocycle.

[0213] In some embodiments of a compound or salt of Formula (III), Head is selected from each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20- membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle and each Rb is independently selected from H and C1-C5 alkyl, s is selected from 0, 1, and 2, each q is selected from 1, 2, 3, and 4. In some embodiments, s is 0. In some embodiments, s is 1. In some embodiments, q is selected from 2 and 3. In some embodiments, q is 2. In some embodiments, 1 is 3. In some embodiments, q is 4. In some embodiments, q is 1. In some embodiments of a compound or salt of Formula (III), Head is selected from independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20- membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle and each w is selected from 1, 2, 3, and 4. In some embodiments, w is each independently selected from 2 and 3. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, w is 4. In some embodiments, w is

[0214] 1.

[0215] In some embodiments of a compound or salt of Formula (III) or (I), RL1Pldis selected

[0216] In some embodiments of a compound or salt of Formula (III) or (I), RL1Pldis selected from the group consisting of is selected from the group consisting of O

[0217] RLipidis selected from the group consisting

[0218] . In some embodiments, Head is selected from

[0219] In some embodiments of a compound or salt of Formula (III), Head is selected from the

[0220] some embodiments, Head is selected from the group consisting of

[0221] In some embodiments of a compound or salt of Formula (III), p is selected from 2 and 4. In some embodiments, p is 2. In some embodiments, p is 4.

[0222] In some embodiments of a compound or salt of Formula (III) or (I), RL1Pldis represented by a structure in TABLE 1 or TABLE 3. In some embodiments of a compound or salt of Formula (III) or (I), RL1Pldis represented by a structure in TABLE 3.

[0223] In some embodiments of a compound or salt of Formula (III), the compound is represented by a structure in TABLE 2 or TABLE 4. In some embodiments of a compound or salt of Formula (III), the compound is represented by a structure in TABLE 4.

[0224] In some aspects, provided herein is a composition comprising a pharmaceutical agent (or therapeutic agent) assembled with a lipid composition. In some embodiments, the lipid composition comprises an ionizable lipid provided herein.

[0225] In some aspects, provided herein is a composition, comprising a pharmaceutical agent assembled with a lipid composition comprising an ionizable lipid represented by Formula (III): Head - (RLipid)P(Formula III) or a pharmaceutically acceptable salt thereof; wherein: wherein each Rais independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each of Rki and Rk3 are independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4and Rks are independently selected from H and C1-C10 alkyl; s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6.

[0226] In some embodiments, the pharmaceutical agent or therapeutic agent comprises a peptide, a protein, or a nucleic acid molecule.

[0227] In some embodiments, the ionizable lipid comprises an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine head group;

[0228] Ri and R2 are each independently selected from C1-C12 alkylene, C1-C12 alkenylene, and 1- to 12- membered heteroalkylene;

[0229] X is , in which each of L3, L4, L5, and Lr, is, independently, a bond, O, S, or NRC; G is O, S, or NRd; Q is ORe, SRf, or NRgRh; r is selected from 1, 2, 3, 4, 5, and 6; and t is t is selected from 1, 2, 3, 4, 5, and 6; each of Rc, Rd, Re, Rf, Rg, and Rhis independently H, Cl -CIO alkyl, 1- to 10- membered heteroalkyl, aryl, or heteroaryl;

[0230] Y and U are each independently a bond, O, S, NR10, or Se; n is 0 or 1 ;

[0231] R3 and R4, are each independently H, Cl -CIO alkyl, 1- to 10-membered heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atom to which they are attached, form =0;

[0232] R5is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20- membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises a nucleic acid molecule (e.g., DNA or RNA) encoding at least one therapeutic peptide. In some embodiments, the pharmaceutical agent can be an mRNA encoding a therapeutic peptide suitable for immunotherapy.

[0233] In some embodiments, n is 0. In some embodiments, n is 1.

[0234] In some embodiments, n is 1 and R3 and R4 together with the atom to which they are attached, form =0. In some embodiments, Y is CH2 and U is O. In some embodiments, Y and U are both O. In some embodiments, U is CH2 and Y is O. In some embodiments, Y and U are NR10. In some embodiments, Y is O and U is NR10. In some embodiments, Y is S and U is NR10.

[0235] In some embodiments, n is 0, and Y and U are both S. In some embodiments, n is 0, and one of Y and U is O. In some embodiments, n is 0, and one of Y and U is Se.

[0236] In some embodiments, Ri is a C1-C12 alkyl, linear or branched. In some embodiments, Ri is a C1-C10 alkyl, linear or branched. In some embodiments, Ri is a C1-C8 alkyl, linear or branched. In some embodiments, Ri is a C1-C6 alkyl, linear or branched. In some embodiments, Ri is a C1-C4 alkyl, linear or branched. In some embodiments, Ri is a C2 alkyl, , ,

[0237] Ri is a 1- to 12-membered heteroalkylene. In some embodiments, R2 is a C1-C12 alkyl, linear or branched. In some embodiments, R2 is a Cl -CIO alkyl, linear or branched. In some embodiments, R2 is a C1-C8 alkyl, linear or branched. In some embodiments, R2 is a C1-C6 alkyl, linear or branched. In some embodiments, R2 is a C1-C4 alkyl, linear or branched. In some embodiments, R l. In some embodiments,

[0238] R2 is a 1- to 12-membered heteroalkylene.

[0239] In some embodiments, the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra”’ are each independently H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C6-C20 aryl, and 6- to 20-membered heteroaryl;, or a RLipid, selected from C1-C20 alkylene, a 1- to 20- membered heteroalkylene, arylene, and heteroarylene.

[0240] In some embodiments, the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein: i) Rbis a substituted or unsubstituted Ci-Ce alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; and ii) nl and n2 are each independently 1, 2, 3, 4, 5, or 6.

[0241] In some embodiments, Rbis a C1-C6 alkyl, linear or branched. In some embodiments, Rbis a substituted C1-C6 alkyl, linear or branched. In some embodiments, a substituent comprises hydroxyl, carbonyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, cyclic amine, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In some embodiments, the substituent comprises hydroxyl, any suitable substituent thereof.

[0242] In some embodiments, nl is 1, 2, 3, 4, 5, or 6. In some embodiments, nl is 2 or 3.

[0243] In some embodiments, n2 is 1, 2, 3, 4, 5, or 6. In some embodiments, n2 is 2 or 3.

[0244] In some embodiments, nl and n2 are identical. In some embodiments, nl and n2 are different.

[0245] In some embodiments, both nl and n2 are 2. In some embodiments, both nl and n2 are

[0246] 3. In some embodiments, both nl and n2 are 4.

[0247] In some embodiments, the amine head group is selected from the group consisting of

[0248] In some embodiments, the at least one hydrophobic tail RLipidhas a structure of wherein each Rki and Rk3 is independently selected from C1-C10 alkyl; each Rk2is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; and each Rk4and Rks is independently selected from H and C1-C10 alkyl.

[0249] In some embodiments, Rki is a C1-C10 alkyl, linear or branched. In some embodiments,

[0250] Rki is a C1-C4 alkyl, linear or branched. In some embodiments, Rki is a C2 alkyl, e.g., In some embodiments, Rki is a C4 alkyl.

[0251] In some embodiments, Rk3 is a C1-C10 alkyl, linear or branched. In some embodiments,

[0252] Rk3 is a C1-C4 alkyl, linear or branched. In some embodiments, Rk3 is a C2 alkyl, e.g., . In some embodiments, Rk3 is a C3 alkyl, e.g., , or . In some embodiments, Rk3 is a C4 alkyl.

[0253] In some embodiments, Rk4 is H. In some embodiments, Rk4 is C1-C10 alkyl. In some embodiments, Rk4 is C1-C4 alkyl. In some embodiments, Rk4 is C4-C10 alkyl.

[0254] In some embodiments, Rks is H. In some embodiments, Rks is C1-C10 alkyl. In some embodiments, Rks is C1-C4 alkyl. In some embodiments, Rks is C4-C10 alkyl.

[0255] In some embodiments, Rk2 is a C1-C20 alkyl. In some embodiments, Rk2 is a C2-C20 alkenyl. In some embodiments, Rk2 is a C2-C20 alkynyl. In some embodiments, Rk2 is a C3- C20 cycloalkyl. In some embodiments, Rk2 is a C1-C20 heteroalkyl. In some embodiments, Rk2 is a 1- to 20-membered heterocycloalkyl, aryl, or heteroaryl. In some embodiments, Rk2 is a C4-C20 alkyl, linear or branched. In some embodiments, Rk2 is a C8-C14 branched alkyl. In some embodiments, Rk2 is a C4-C20 alkenyl, linear or branched. In some embodiments, Rk2 is a C8-C14 branched alkenyl.

[0256] In some embodiments, the at least one hydrophobic tail is selected from the TABLE 1.

[0257] TABLE 1. Exemplary Hydrophobic Tail

[0258] In some embodiments, the at least one hydrophobic tail is selected from the TABLE 3.

[0259] TABLE 3. Exemplary Hydrophobic Tail

[0260] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails. In some embodiments, the at least two hydrophobic tails are independently of structure . In some embodiments, the at least two hydrophobic tails are identical. In some embodiments, the at least two hydrophobic tails are not identical. In some embodiments, one of the at least two hydrophobic tails is different from the rest.

[0261] In some embodiments, the lipid composition comprises at least three hydrophobic tails. In some embodiments, the at least three hydrophobic tails are independently of structure . In some embodiments, the at least three hydrophobic tails are identical. In some embodiments, the at least three hydrophobic tails are not identical. In some embodiments, one of the at least three hydrophobic tails is different from the rest.

[0262] In some embodiments, the lipid composition comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are independently of structure . In some embodiments, the two hydrophobic tails are identical. In some embodiments, the two hydrophobic tails are not identical.

[0263] In some embodiments, the lipid composition comprises three hydrophobic tails. In some embodiments, the three hydrophobic tails are independently of structure jn someembodiments, the three hydrophobic tails are identical. In some embodiments, two of the three hydrophobic tails are identical, and the third hydrophobic tail is different. In some embodiments, all three hydrophobic tails are different.

[0264] In some embodiments, the lipid composition comprises four hydrophobic tails. In some embodiments, the four hydrophobic tails are independently of structure . in some embodiments, the four hydrophobic tails are identical. In some embodiments, three of the four hydrophobic tails are identical, and the fourth hydrophobic tail is different. In some embodiments, two of the four hydrophobic tails are identical, the other two hydrophobic tails are identical, and the two are different from other two. In some embodiments, two of the four hydrophobic tails are identical while the other two are different from each other and are different from the two. In some embodiments, all four hydrophobic tails are different.

[0265] In some embodiments, the ionizable lipid is selected from TABLE 2.

[0266]

[0267]

[0268] Ill

[0269]

[0270]

[0271] In some embodiments, the ionizable lipid is selected from TABLE 4.

[0272]

[0273] In some embodiments, the composition provided herein further comprises a steroid. In some embodiments, the steroid comprises a cholesterol or a cholesterol derivative. In some embodiments, the composition provided herein further comprises a helper lipid. In some embodiments, the helper lipid comprises l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the composition provided herein further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some embodiments, the lipid composition further comprises a modification comprising a cellpenetrating peptide. In some cases, the cell-penetrating peptide is transactivator of transcription (TAT).

[0274] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid, a helper lipid, and a polymer conjugated lipid.

[0275] In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 80%, about 10% to about 90%, from about 20% to about 30%, from about 20% to about 30%, from about 20% to about 40%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 30% to about 40%, from about 30% to about 50%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about 80%, from about 30% to about 90%, from about 40% to about 50%, from about 40% to about 60%, from about 40% to about 70%, from about 40% to about 80%, from about 40% to about 90%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, from about 70% to about 80%, from about 70% to about 90%, or from about 80% to about 90%.

[0276] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0277] In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.

[0278] In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0279] In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 14 / 4 / 1 / 1, about 15 / 4 / 1 / 1, about 16 / 4 / 1 / 1, about 17 / 4 / 1 / 1, about 18 / 4 / 1 / 1, about 19 / 4 / 1 / 1, about 20 / 4 / 1 / 1, about 14 / 4 / 2 / 1, about 15 / 4 / 2 / 1, about 16 / 4 / 2 / 1, about 16.4 / 4 / 2 / 1, about 16.6 / 4 / 2 / 1, about 16.8 / 4 / 2 / 1, about 17 / 4 / 2 / 1, about 18 / 4 / 2 / 1, about 19 / 4 / 2 / 1, or about 20 / 4 / 2 / 1. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.4 / 4 / 2 / 1 or 16.6 / 4 / 2 / 1.

[0280] In some embodiments, the lipid composition comprises an ionizable lipid disclosed in this application, a steroid and a helper lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage of from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 50% to about 80%, from about 60% to about 80%, or from about 70% to about 80%.

[0281] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage of from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 7.5% to about 40%, from about 7.5% to about 30%, from about 7.5% to about 20%, from about 7.5% to about 15%, from about 7.5% to about 10%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, from about 10% to about 17.5%, or from about 10% to about 15%. In some embodiments, the helper lipid is present in the lipid composition at a weight percentage of from about 12% to about 14%.

[0282] In some embodiments, the steroid is present in the lipid composition at a weight percentage of from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 7.5% to about 40%, from about 7.5% to about 30%, from about 7.5% to about 20%, from about 7.5% to about 15%, from about 7.5% to about 10%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 20%, from about 10% to about 17.5%, from about 15% to about 30%, from about 15% to about 25%, from about 15% to about 22.5%, from about 15% to about 20%, or from about 15% to about 17.5%. In some embodiments, the steroid is present in the lipid composition at a weight percentage of from about 16.5% to about 17.5%.

[0283] In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid is about 1 / 1 / 1, 2 / 1 / 1, about 3 / 1 / 1, about 4 / 1 / 1, about 5 / 1 / 1, about 6 / 1 / 1, about 2 / 2 / 1, about 3 / 2 / 1, about 4 / 2 / 1, about 5 / 2 / 1, about 20 / 4 / 3, about 17 / 4 / 3, about 16 / 4 / 3, about 8 / 2 / 1, or about 6 / 2 / 1. In some embodiments, the weight ratio of the ionizable lipid / steroid / helper lipid is about 16.6 / 4 / 3.

[0284] In some embodiments, the lipid composition further comprises an excipient. The excipient can comprise EC- 16, (2-hydroxypropyl)-P-cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, or Bile salts. In some embodiments, the excipient is present in the composition at a weight percentage from about 5% to about 60%. In some embodiments, the excipient is present in the composition at a weight percentage from about 1% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.

[0285] In some embodiments, the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof. In some embodiments, the polynucleotide is a messenger ribonucleic acid (mRNA).

[0286] In some embodiments, the pharmaceutical agent comprises a polynucleotide that encodes or is configured to regulate a target gene or a gene product thereof.

[0287] In some embodiments, the pharmaceutical agent comprises (a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or (b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof.

[0288] In some embodiments, the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid.

[0289] In some embodiments, the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease. In some embodiments, the heterologous endonuclease comprises a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease. In some embodiments, the polynucleotide comprising a sequence that encodes the heterologous endonuclease.

[0290] In some embodiments, the pharmaceutical agent (or therapeutic agent) comprises mRNA encoding at least one therapeutic peptide for immunotherapy. In some embodiments, the pharmaceutical agent (or therapeutic agent) is suitable for cancer immunotherapy. In some embodiments, the pharmaceutical agent is assembled in the lipid composition at a weight ratio of the pharmaceutical agent / lipid composition of from about 1:200 to about 1:100, from about 1:200 to about 1:50, from about 1:200 to about 1:40, from about 1:200 to about 1:30, from about 1:200 to about 1:20, from about 1:200 to about 1:10, from about 1:200 to about 1:5, from about 1:200 to about 1:1, from about 1:100 to about 1:50, from about 1:100 to about 1:40, from about 1:100 to about 1:25, from about 1:100 to about 1:20, from about 1:100 to about 1:15, from about 1:100 to about 1:10, from about 1:100 to about 1:5, from about 1:100 to about 1:1, from about 1:50 to about 1:30, from about 1:50 to about 1:25, from about 1:50 to about 1:20, from about 1:40 to about 1:30, from about 1:40 to about 1:25, from about 1:40 to about 1:20, or from about 1:30 to 1:20. In some embodiments, the pharmaceutical agent is assembled in the lipid composition at a weight ratio of the pharmaceutical agent / lipid composition of 1:25.

[0291] In some embodiments, the target gene or the gene product thereof is specific to or primarily found in a target organ (e.g., liver, spleen, or lymph nodes) or a target cell (e.g., a immune cell provided herein; e.g., a T cell) of a subject.

[0292] In some embodiments, the target gene or the gene product thereof is associated with a disease or disorder of the target organ or the target cell.

[0293] In some embodiments, the gene modulating moiety is configured to provide a modified expression profile of the target gene or the gene product thereof in a target organ (e.g., liver, spleen, lymph nodes) or a target cell (e.g., an immune cell provided herein, e.g., a T cell) of a subject.

[0294] In some embodiments, the composition is formulated for systemic or local administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for intramuscular administration.

[0295] In some embodiments, the composition provided herein can preferentially deliver a therapeutic payload to a target organ. In some embodiments, the target organ comprises a liver, spleen, lymph nodes, brain, kidney, heart, or lung. In some embodiments, the target organ is a liver. In some embodiments, the target organ comprises secondary lymphoid tissue. In some embodiments, the target organ is a spleen. In some embodiments, the target organ is a lymph node.

[0296] In some embodiments, the composition provided herein can preferentially deliver a therapeutic payload to a target cell of a target organ. In some embodiments, the target cell comprises a cell of a liver, spleen, lymph nodes, brain, kidney, heart, or lung. In some embodiments, the target cell is a cell of the liver. In some embodiments, the target cell comprises a cell within secondary lymphoid tissue. In some embodiments, the target cell is a cell of the spleen. In some embodiments, the target cell comprises a lymphocyte. In some embodiments, the target cell comprises a T cell, B cell, macrophage, or dendritic cell. In some embodiments, the target cell comprises an antigen presenting cell (APC).

[0297] Additional Lipids

[0298] In some embodiments, the lipid composition further comprises an additional lipid comprising a steroid or a steroid derivative, a PEG lipid, and a helper lipid (e.g., phospholipids or other zwitterionic lipids).

[0299] In some embodiments, the lipid composition further comprises a helper lipid. In some embodiments, the helper lipid comprises a lipid that contributes to the stability or delivery efficiency of the lipid compositions. In some embodiments, the helper lipid comprises a zwitterionic lipid. In some embodiments, the helper lipid comprises a phospholipid. In some embodiments, the phospholipid may contain one or two long chain (e.g., C6-C24) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid- like molecules with both a positive charge and a negative charge. In some embodiments of the lipid compositions, the phospholipid is not an ethylphosphocholine. In some embodiments, the helper lipid can comprise l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0300] In some embodiments, the compositions may further comprise a molar percentage of the phospholipid to the total lipid composition from about 5 to about 30.

[0301] In some embodiments, the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0302] In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 23%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 12% to about 18%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage of at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 23%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage of at most about 8%, at most about 10%, at most about 12%, at most about 15%, at most about 18%, at most about 20%, or at most about 23%.

[0303] In some embodiments, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula: . In some embodiments, a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as: some embodiments, the steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by the formula: described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system. In some embodiments, the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both a cholesterol and a sterol or a derivative thereof.

[0304] In some embodiments, the compositions may further comprise a molar percentage of the steroid to the total lipid composition from about 20 to about 60. In some embodiments, the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.

[0305] In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 15% to about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 25% to about 35%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 30% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 30%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage of at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 46%.

[0306] In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3 -amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-.sn-glyccrol. In some embodiments, the PEG modification is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000. Some non-limiting examples of lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0307] In some embodiments, the PEG lipid has a structural formula: , wherein: R12 and R13 are each independently alkyl(c<24), alkenyl(c<24), or a substituted version of either of these groups; Reis hydrogen, alkyl(c<8), or substituted alkyl(c<8>; and x is 1-250. In some embodiments, Reis alkyl(c<8) such as methyl. R12 and R13 are each independently alkyl(c<4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0308] In some embodiments, the PEG lipid has a structural formula: , wherein: m is an integer between 1 and 100 and n2 and m are each independently selected from an integer between 1 and 29. In some embodiments, m is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, m is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, m is from 5 to 23. In some embodiments, m is 11 to about 17.

[0309] In some embodiments, the polymer conjugated lipid comprises 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).

[0310] In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 20%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1% to about 8%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 2% to about 7%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, or at least about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 2.5%, at most about 3%, at most about 3.5%, at most about 4%, at most about 4.5%, at most about 5%, at most about 5.5%, at most about 6%, at most about 6.5%, at most about 7%, at most about 7.5%, at most about 8%, at most about 8.5%, at most about 9%, at most about 9.5%, at most about 10%, at most about 15%, or at most 20%.

[0311] In some embodiments, the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.

[0312] Therapeutic Payloads

[0313] Provided herein are lipid nanoparticle compositions comprising a therapeutic payload (e.g., a pharmaceutical agent or a therapeutic agent). The lipids and lipid compositions can be used for delivering a plurality of therapeutic payloads. In some embodiments, the therapeutic payload can comprise a peptide, a nucleic acid molecule, a small molecule drug, or a combination thereof. In some embodiments, the therapeutic payload comprises a therapeutic peptide. In some embodiments, the therapeutic payload comprises a nucleic acid molecule. The nucleic acid molecule can comprise an engineered DNA, RNA (e.g., mRNA, sgRNA), a hybrid nucleic acid or a combination thereof. In some embodiments, the nucleic acid molecule can encode a therapeutic peptide. In some embodiments, the lipid nanoparticle (LNP) compositions comprise a therapeutic peptide or nucleic acid molecules (e.g., mRNA or DNA) encoding a therapeutic peptide. In some embodiments, the LNP comprises an mRNA encoding a therapeutic peptide. The therapeutic peptides may be suitable for immunotherapy. In some embodiments, the therapeutic peptide comprises a secretary peptide. In some embodiments, the therapeutic peptide comprises a transmembrane peptide. In some embodiments, the therapeutic peptide comprises an intracellular peptide.

[0314] In some cases, the therapeutic peptides for immunotherapy comprise heterologous immune receptors (e.g., antigen receptors), antigen-binding peptides (e.g., antibodies or fragments thereof), or immunomodulators. In some cases, a heterologous immune receptor comprises an antigen receptor, e.g., a chimeric antigen receptor (CAR) on immune cells (e.g., T cells). CARs can be designed for cell-based immunotherapy, for example, for CAR-T or CAR-pool therapy. The antigen-binding peptides of the present disclosure comprise secreted therapeutic peptides that can bind to an antigen (e.g., a cancer antigen). In some cases, the antigen-binding peptides can be multi- specific and bind to more than one antigen. In some embodiments, the multi- specific antigen-binding peptide can bind to an immune cell marker (e.g., CD3 on a T-cell) and a tumor antigen. In some embodiments, the multi- specific antigenbinding peptide comprises a bispecific T cell engager (BiTE). In some cases, the therapeutic peptides may further comprise a protein tag (e.g., a 6X His tag). In some cases, the protein tag can facilitate identification or purification of the therapeutic peptide.

[0315] In some cases, the LNPs of the present disclosure comprises one or more therapeutic peptides. In some embodiments, the LNP comprises two or more therapeutic peptides. In some embodiments, the LNP comprises 1, 2, 3, 4, 5, 6 or more therapeutic peptides. In some embodiments, the LNP comprises one or more nucleic acid molecules (e.g., mRNA or DNA) encoding one or more therapeutic peptides for immunotherapy. In some cases, the LNPs comprises mRNA(s) encoding 1, 2, 3, 4, 5, 6, or more therapeutic peptides. In some embodiments, the LNP comprises a therapeutic peptide and a nucleic acid molecule encoding a therapeutic peptide.

[0316] In some embodiments, the therapeutic peptides may be an antigen-binding peptide. In some embodiments, the therapeutic peptides may be a multispecific antigen-binding peptide. In some cases, a multispecific antigen-binding peptide comprises a first domain that binds to an antigen expressed by an immune cell and a second domain that binds to an antigen expressed by a target cell (e.g., a tumor cell). The multispecific antigen-binding peptide can bind to an antigen expressed by any one of the immune cells provided herein. In some embodiments, the therapeutic peptide comprises a chimeric antigen receptor derived from a neutralizing antibody comprising an antigen-binding fragment, a transmembrane domain, and an intracellular signaling domain described herein. The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0317] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity and helper activity, including the secretion of cytokines. In some cases, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some cases, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP 12.

[0318] The term “co stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that can be used for an efficient immune response. Costimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la / CD18) and 4-1BB (CD137).

[0319] A costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4- IBB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0320] The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0321] With respect to the transmembrane domain, in various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In some cases, the transmembrane domain is capable of homodimerization with another CAR on the CAR-T cell surface. In some cases, the amino acid sequence of the transmembrane domain can be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR- T cell.

[0322] The transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some cases, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. In some cases, the transmembrane domain can include at least the transmembrane region(s) of e.g., the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0323] In some cases, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge or a spacer, e.g., a hinge from a human protein. In some cases, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In one aspect, the hinge or spacer comprises an IgG4 hinge. The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0324] Examples of intracellular signaling domains for use in the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0325] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or costimulatory signal can also be involved. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling domain, e.g., a co stimulatory domain).

[0326] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0327] Examples of ITAM containing primary intracellular signaling domains that are of particular use herein include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some cases, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain, of CD3-zeta. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0328] A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that can play a role for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3) :696-706).

[0329] A short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequence. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[0330] In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some cases, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In some cases, the intracellular signaling domain comprises two costimulatory signaling domains. In some cases, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0331] In some embodiments, the therapeutic peptide can comprise an immunomodulator or a nucleic acid molecule (e.g., mRNA) encoding an immunomodulator. In some cases, an immunomodulator comprises a cytokine, cytokine receptor, chemokine, chemokine receptor, immune co-receptor, or immune co-receptor ligand. Expression of the immunomodulator can be driven by an expression regulatory region disclosed herein.

[0332] In some cases, the therapeutic peptide comprises a cytokine or a functional fragment thereof, for example, G-CSF, GITRL, GM-CSF, IFN-a, IFN-P, IFN-y, IL-IRA, IL-la, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 18, IL-20, IL-23, LIF, LIGHT, LT-p, M-CSF, MSP, OSM, OX40L, SCF, TALL-1, TGF-p, TGF-pi, TGF-P2, TGF-P3, TNF-a, TNF-p, TRAIL, TRANCE, or TWEAK. In some cases, the therapeutic peptide comprises a cytokine receptor or a functional fragment thereof, for example, a common gamma chain receptor, a common beta chain receptor, an interferon receptor, a TNF family receptor, a TGF-B receptor, Apo3, CD 114, CD115, CD116, CD117, CD118, CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD124, CD126, CD127, CD130, CD131, CD132, CD212, CD213, CD213al, CD213al3, CD213a2, CD25, CD27, CD30, CD4, CD40, CD95 (Fas), CDwl l9, CDwl21b, CDwl25, CDwl31, CDwl36, CDwl37 (41BB), CDw210, CDw217, GITR, HVEM, IL-11R, IL-l lRa, IL-14R, IL-15R, IL-15Ra, IL-18R, IL-18Ra, IL-18RP, IL-20R, IL-20Ra, IL-20RP, IL-9R, LIFR, LTpR, OPG, OSMR, 0X40, RANK, TACI, TGF-PR1, TGF-PR2, TGF-PR3, TRAILR1, TRAILR2, TRAILR3, or TRAILR4.

[0333] In some cases, the therapeutic peptide for immunotherapy comprises a chemokine or a functional fragment thereof, for example, ACT-2, AMAC-a, ATAC, ATAC, BLC, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL3, CCL4, CCL5, CCL7, CCL8, CKb- 6, CKb-8, CTACK, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, DC-CK1, ELC, ENA- 78, eotaxin, eotaxin-2, eotaxin-3, Eskine, exodus-1, exodus-2, exodus-3, fractalkine, GCP-2, GROa, GROb, GROg, HCC-1, HCC-2, HCC-4, 1-309, IL-8, ILC, IP-10, I-TAC, LAG-1, LARC, LCC-1, LD78a, LEC, Lkn-1, LMC, lymphoactin, lymphoactin b, MCAF, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MDNCF, MGSA-a, MGSA-b, MGSA-g, Mig, MIP-ld, MIP- la, MIP-ip, MIP-2a, MIP-2b, MIP-3, MIP-3a, MIP-3P, MIP-4, MIP-4a, MIP-5, MPIF-1, MPIF-2, NAF, NAP-1, NAP-2, oncostatin, PARC, PF4, PPBP, RANTES, SCM-la, SCM-lb, SDF-la / p, SLC, STCP-1, TARC, TECK, XCL1, or XCL2.

[0334] In some cases, the therapeutic peptide for immunotherapy comprises a chemokine receptor or a functional fragment thereof, for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, XCRl, or XCR1.

[0335] In some cases, the therapeutic peptide for immunotherapy comprises costimulatory immune receptor, or a functional fragment thereof, for example, CD28, 2B4 (CD244, SLAMF4), 4-1BB (CD137), CD2 (LFA2, 0X34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM (CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LTpR (TNFRSF3), LylO8 (NTBA,CD352,SLAMF6), Ly9 (CD229,SLAMF3), 0X40 (CD134), SLAM (CD15O,SLAMF1), TIM1 (HAVCR1,KIM1), or TIM2.

[0336] In some cases, therapeutic peptide for immunotherapy comprises an activating NK receptor, or a functional fragment thereof, for example, CD100 (SEMA4D), CD16 (FcgRIIIA), CD160 (BY55), CD244 (2B4, SLAMF4), CD27, CD94- NKG2C, CD94- NKG2E, CD94- NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226), KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49, NCR, NKG2D (KLRK1, CD314), NKp30 (NCR3), NKp44 (NCR2), NKp46 (NCR1), NKp80 (KLRF1, CLEC5C), NTB-A (SLAMF6), PSGL1, or SLAMF7 (CRACC, CS1, CD319).

[0337] In some cases, the therapeutic peptide for immunotherapy comprises an Fey receptor (FcyR), an Fes receptor (FcsR), an Fea receptor (FcaR), an Fcp receptor (FcpR), neonatal Fc receptor (FcRn), CD4, CD5, CD8, CD21, CD22, CD27, CD28, CD32, CD40, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (ICOS), CD247^, CD247r|, 41BB, DAP10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NFAT, NF-KB, PLC-y, iC3b, C3dg, C3d, Zap70, MyD88, a functional fragment thereof, or a combination thereof.

[0338] In some cases, the therapeutic peptide for immunotherapy comprises a domain that is, is derived from, interacts with, increases expression of, or activates a transcription factor, such as, for example, E2A, Pax5, EBF, PU.l, Ikaros, GATA3, Th-POK, Tbet, Bcl6, NF-KB, NFAT, AP-1, NFAT, STAT1, STAT2, STAT3, STAT4, STAT5, STAT5A, STAT5B, STAT6, STAT7, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, AP-1, Eomes, FoxP3, Id2, PLZF, ROR-gamma-T, TCF7, ThPOK, or any combination thereof.

[0339] Therapeutic Peptides in Immunotherapy

[0340] Provided herein are lipid nanoparticle compositions comprising nucleic acid molecules (e.g., mRNA) encoding therapeutic peptides for immunotherapy. In some embodiments, the mRNA encapsulated by the lipid composition provided herein comprises sequences that encode a therapeutic peptide for immunotherapy. In some cases, the therapeutic peptides for immunotherapy comprise heterologous immune receptors (e.g., antigen receptors), antigenbinding peptides (e.g., antibodies or fragments thereof), or immunomodulators. In some cases, a heterologous immune receptor comprises an antigen receptor, e.g., a chimeric antigen receptor (CAR) on immune cells (e.g., T cells). CARs can be designed for cell-based immunotherapy, for example, for CAR-T or CAR-pool therapy. The antigen-binding peptides of the present disclosure comprise secreted therapeutic peptides that can bind to an antigen (e.g., a cancer antigen). In some cases, the antigen-binding peptides can be multi- specific and bind to more than one antigen. The multi- specific antigen-binding peptide can bind to an immune cell marker (e.g., CD3 on a T-cell) and a tumor antigen.

[0341] In some cases, the therapeutic peptides encoded by the mRNA encapsulated by the LNPs of the present disclosure comprise a protein tag (e.g., a 6X His tag). In some cases, the protein tag can facilitate identification or purification of the therapeutic peptide.

[0342] In some cases, more than one therapeutic peptide can be encoded by the mRNA encapsulated by the LNPs of the present disclosure. In some cases, the LNPs of the present disclosure comprises mRNA encoding 1, 2, 3, 4, 5, 6, or more therapeutic peptides.

[0343] In some embodiments, the LNPs of the disclosure comprises mRNAs that encode a multispecific antigen-binding peptide for immunotherapy. In some cases, a multispecific antigen-binding peptide comprises a first domain that binds to an antigen expressed by an immune cell and a second domain that binds to an antigen expressed by a tumor cell. The multispecific antigen-binding peptide can bind to an antigen expressed by any one of the immune cells provided herein.

[0344] In some embodiments, the LNPs of the disclosure comprises mRNAs that encode a chimeric antigen receptor derived from a neutralizing antibody comprising an antigen-binding fragment, a transmembrane domain, and an intracellular signaling domain described herein. The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0345] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell. Examples of immune effector function, e.g., in a CAR-T cell, include cytolytic activity and helper activity, including the secretion of cytokines. In some cases, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some cases, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP 12.

[0346] The term “co stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that can be used for an efficient immune response. Costimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la / CD18) and 4-1BB (CD137).

[0347] A costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4- IBB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0348] The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0349] With respect to the transmembrane domain, in various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In some cases, the transmembrane domain is capable of homodimerization with another CAR on the CAR-T cell surface. In some cases, the amino acid sequence of the transmembrane domain can be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR- T cell.

[0350] The transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some cases, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. In some cases, the transmembrane domain can include at least the transmembrane region(s) of e.g., the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0351] In some cases, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge or a spacer, e.g., a hinge from a human protein. In some cases , the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In one aspect, the hinge or spacer comprises an IgG4 hinge.

[0352] The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0353] Examples of intracellular signaling domains for use in the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[0354] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or costimulatory signal can also be involved. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling domain, e.g., a co stimulatory domain).

[0355] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0356] Examples of ITAM containing primary intracellular signaling domains that are of particular use herein include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some cases, a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain, of CD3-zeta. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[0357] A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that can play a role for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (EFA-1), CD2, CD7, EIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3) :696-706).

[0358] A short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequence. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[0359] In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some cases, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In some cases, the intracellular signaling domain comprises two costimulatory signaling domains. In some cases, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0360] In some embodiments, the mRNA-encoded therapeutic peptides for immunotherapy provided herein comprise immunomodulators. In some cases, an immunomodulator comprises a cytokine, cytokine receptor, chemokine, chemokine receptor, immune co-receptor, or immune co-receptor ligand. Expression of the immunomodulator can be driven by an expression regulatory region disclosed herein.

[0361] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises a cytokine or a functional fragment thereof, for example, G-CSF, GITRL, GM-CSF, IFN-a, IFN-P, IFN-y, IE-1RA, IF-la, IL-ip, IE-2, IE-3, IE-4, IE-5, IE-6, IL-7, IL-9, IL-10, IL-11, IL- 12, IL- 13, IL- 14, IL- 15, IL- 16, IL- 17, IL- 18, IL-20, IL-23, LIF, LIGHT, LT-p, M-CSF, MSP, OSM, OX40L, SCF, TALL-1, TGF-p, TGF-pi, TGF-P2, TGF-P3, TNF-a, TNF-p, TRAIL, TRANCE, or TWEAK.

[0362] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises a cytokine receptor or a functional fragment thereof, for example, a common gamma chain receptor, a common beta chain receptor, an interferon receptor, a TNF family receptor, a TGF- B receptor, Apo3, CD114, CD115, CD116, CD117, CD118, CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD124, CD126, CD127, CD130, CD131, CD132, CD212, CD213, CD213al, CD213al3, CD213a2, CD25, CD27, CD30, CD4, CD40, CD95 (Fas), CDwl l9, CDwl21b, CDwl25, CDwl31, CDwl36, CDwl37 (41BB), CDw210, CDw217, GITR, HVEM, IL-11R, IL-l lRa, IL-14R, IL-15R, IL-15Ra, IL-18R, IL-18Ra, IL- 18RP, IL-20R, IL-20Ra, IL-20RP, IL-9R, LIFR, LTpR, OPG, OSMR, 0X40, RANK, TACI, TGF-PR1, TGF-PR2, TGF-PR3, TRAILR1, TRAILR2, TRAILR3, or TRAILR4.

[0363] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises a chemokine or a functional fragment thereof, for example, ACT-2, AMAC-a, ATAC, ATAC, BLC, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL3, CCL4, CCL5, CCL7, CCL8, CKb-6, CKb-8, CTACK, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, DC-CK1, ELC, ENA-78, eotaxin, eotaxin-2, eotaxin-3, Eskine, exodus-1, exodus-2, exodus-3, fractalkine, GCP-2, GROa, GROb, GROg, HCC-1, HCC-2, HCC-4, 1-309, IL-8, ILC, IP- 10, LTAC, LAG-1, LARC, LCC-1, LD78a, LEC, Lkn-1, LMC, lymphoactin, lymphoactin b, MCAF, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MDNCF, MGSA-a, MGSA-b, MGSA-g, Mig, MIP-ld, MIP-la, MIP-ip, MIP-2a, MIP-2b, MIP-3, MIP-3a, MIP-3P, MIP-4, MIP-4a, MIP-5, MPIF-1, MPIF-2, NAF, NAP-1, NAP-2, oncostatin, PARC, PF4, PPBP, RANTES, SCM-la, SCM-lb, SDF-la / p, SLC, STCP-1, TARC, TECK, XCL1, or XCL2.

[0364] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises a chemokine receptor or a functional fragment thereof, for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, XCR1, or XCR1.

[0365] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises costimulatory immune receptor, or a functional fragment thereof, for example, CD28, 2B4 (CD244, SLAMF4), 4-1BB (CD137), CD2 (LFA2, 0X34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM (CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LTpR (TNFRSF3), LylO8 (NTBA,CD352,SLAMF6), Ly9 (CD229,SLAMF3), 0X40 (CD134), SLAM (CD15O,SLAMF1), TIM1 (HAVCR1,KIM1), or TIM2.

[0366] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises an activating NK receptor, or a functional fragment thereof, for example, CD 100 (SEMA4D), CD16 (FcgRIIIA), CD160 (BY55), CD244 (2B4, SLAMF4), CD27, CD94- NKG2C, CD94- NKG2E, CD94-NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226), KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49, NCR, NKG2D (KLRK1, CD314), NKp30 (NCR3), NKp44 (NCR2), NKp46 (NCR1), NKp80 (KLRF1, CLEC5C), NTB-A (SLAMF6), PSGL1, or SLAMF7 (CRACC, CS1, CD319).

[0367] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises an Fey receptor (FcyR), an Fes receptor (FcsR), an Fea receptor (FcaR), an Fcp receptor (FcpR), neonatal Fc receptor (FcRn), CD4, CD5, CD8, CD21, CD22, CD27, CD28, CD32, CD40, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (ICOS), CD247^, CD247r|, 41BB, DAP10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NFAT, NF-KB, PLC-y, iC3b, C3dg, C3d, Zap70, MyD88, a functional fragment thereof, or a combination thereof.

[0368] In some cases, the mRNA-encoded therapeutic peptides for immunotherapy comprises a domain that is, is derived from, interacts with, increases expression of, or activates a transcription factor, such as, for example, E2A, Pax5, EBF, PU.l, Ikaros, GATA3, Th-POK, Tbet, Bcl6, NF-KB, NFAT, AP-1, NFAT, STAT1, STAT2, STAT3, STAT4, STAT5, STAT5A, STAT5B, STAT6, STAT7, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, AP-1, Eomes, FoxP3, Id2, PLZF, ROR-gamma-T, TCF7, ThPOK, or any combination thereof.

[0369] Nucleic acids

[0370] The LNPs provided herein comprise nucleic acid molecules (e.g., mRNA) comprising an expression region that encodes a therapeutic peptide for immunotherapy provided herein. In some cases, the mRNA encapsulated by the lipid composition provided herein further comprises regulatory sequences that can facilitate and / or promote expression of the therapeutic peptide for immunotherapy. The regulatory sequences can comprise a 5’ cap, a 5’ untranslated region, a promoter, a signal peptide sequence, a 3’ untranslated region, and a poly- A tail. In some cases, the regulatory sequences comprise an enhancer (e.g., CMV enhancer) to further enhance expression of the therapeutic peptide.

[0371] In some cases, the mRNA encodes a therapeutic peptide for immunotherapy comprising a signal peptide and the signal peptide can be cleaved during post-translational processing.

[0372] In some cases, the mRNA encapsulated by the LNPs of the present disclosure comprises a naturally occurring or an artificial promoter. In some cases, the mRNA comprises a promoter that is specific for expression in immune cells as compared to non-immune cells. In some cases, the mRNA comprises a T-cell specific promoter and the LNPs comprising the mRNA encoding a therapeutic peptide can be used for CAR-T therapy. In some cases, the T-cell specific promoter comprises promoters that drive endogenous expression of T-cell specific proteins comprising CD3 (e.g., CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta), CD4, CD8, CD28, TCRB or TRAC. In some cases, the promoter can result in stronger expression of therapeutic peptides in immune cells comprising lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alphabeta T cells, gamma-delta T cells, T regulatory cells (Tregs), cytotoxic T lymphocytes, Thl cells, Th2 cells, Thl7 cells, Th9 cells, naive T cells, memory T cells, effector T cells, effectormemory T cells (TEM), central memory T cells (TCM), resident memory T cells (TRM), follicular helper T cells (TFH), Natural killer T cells (NKTs), tumor-infiltrating lymphocytes (TILs), Natural killer cells (NKs), Innate Lymphoid Cells (ILCs), ILC1 cells, ILC2 cells, ILC3 cells, lymphoid tissue inducer (LTi) cells, B cells, Bl cells, Bia cells, Bib cells, B2 cells, plasma cells, B regulatory cells, memory B cells, marginal zone B cells, follicular B cells, germinal center B cells, antigen presenting cells (APCs), monocytes, macrophages, Ml macrophages, M2 macrophages, tissue-associated macrophages, dendritic cells, plasmacytoid dendritic cells, neutrophils, mast cells, basophils, eosinophils, common myeloid progenitors, common lymphoid progenitors, or any combination thereof.

[0373] In some cases, the mRNA comprises a hepatocyte- specific promoter and the LNPs comprising the mRNA encoding a secreted therapeutic peptide (e.g., an antigen-binding fragment). In some cases, the mRNA comprises a strong promoter that enhances the expression of the therapeutic peptide for immunotherapy. The strong promoter can comprise a Human cytomegalovirus (hCMV), chicken beta-actin / CMV enhancer (CAG), elongation factor- 1 alpha (EFla), or phosphoglycerokinase (PGK) promoter.

[0374] In some cases, an mRNA encoding a therapeutic peptide for immunotherapy disclosed herein comprises natural, synthetic, and / or artificial nucleotide analogues or bases. In some cases, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of a deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0375] In some cases, a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Illustrative alkyl moiety includes, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[0376] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2’-0-M0E) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety whereas the 2'0-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.

[0377] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen. In some instances, this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.

[0378] In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxy- methylene-linked bicyclic ribonucleotide monomer.

[0379] In some cases, additional modifications at the 2' hydroxyl group include 2'-deoxy, T- deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'- O-dimethylaminopropyl (2'-0-DMAP), T-O- dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'-O-N-methylacetamido (2'-0-NMA).

[0380] In some cases, a nucleotide analogue comprises a modified base, for example, Nl- methylpseudouridine, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6- methylguanine, N, N, -dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1- methy lino sine, 3-methyluridine, 5-methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5- halouridine, 4-acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3-methylcytidine, 6- methyluridine, 2- methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (such as 7-deaza- adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5- oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties, in some cases are or are based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes universal bases. By way of example, universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.

[0381] In some cases, one or more modifications optionally occur at the internucleotide linkage. In some instances, a modified intemucleotide linkages can include, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5'- alkylenephosphonates; 5'-methylphosphonate; 3'-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3'-5'linkage or 2'-5'linkage; phospho triesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates ; arylphosphonothioates ; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3'- alkylphosphoramidates; aminoalky Iphosphoramidates; thionophosphoramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazos; methylenedimethylhydrazos; formacetals; thioformacetals; oximes; methyleneiminos; methylenemethylimino s; thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silyl or siloxane linkages; alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that are saturated or unsaturated and / or substituted and / or contain heteroatoms; linkages with morpholino structures, amides, or polyamides wherein the bases are attached to the aza nitrogens of the backbone directly or indirectly; and combinations thereof.

[0382] In some cases, one or more modifications comprise a modified phosphate backbone in which the modification generates a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation to generate an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1, 1 - dimethylbutyl, 2,2-dimethylbutyl, 3.3- dimethylbutyl, and 2-ethylbutyl groups. In some cases, a modified phosphate is a phosphate group as described in U.S. Patent No. 9481905.

[0383] In some embodiments, additional modified phosphate backbones comprise methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethylphosphonate. In some cases, the modified phosphate is methylthiophosphonate. In some cases, the modified phosphate is methoxyphosphonate.

[0384] In some cases, one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3' or the 5' terminus. For example, the 3' terminus optionally include a 3' cationic group, or by inverting the nucleoside at the 3 '-terminus with a 3 '-3' linkage. In another alternative, the 3'-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3' C5- aminoalkyl dT. In an additional alternative, the 3 '-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5 '-terminus is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some cases, the 5'-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

[0385] Methods

[0386] Provided herein are methods of treating a disease (e.g., a cancer) using the lipid nanoparticle (LNP) compositions described herein, wherein the LNPs comprise nucleic acid molecules (e.g., mRNA) encoding one or more therapeutic peptides (e.g., antigen-binding peptide or immunomodulator) for immunotherapy. In some embodiments of the method, the composition (or pharmaceutical composition) of the present application can be administrated through any suitable routes comprising parenteral delivery (e.g., injections), such as intravenous, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

[0387] In some embodiments of the method, the pharmaceutical composition of the present application can be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted.

[0388] In some embodiments, the composition of the present application can be injected into the site of injury, disease manifestation, or pain, for example. In some embodiments, the composition of the present application can be provided in lozenges for oral, tracheal, or esophageal application. In some embodiments, the composition of the present application can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines. In some embodiments, the composition of the present application can be supplied in suppository form for rectal or vaginal application. In some embodiments, the composition of the present application can even be delivered to the eye by use of creams, drops, or even injection.

[0389] In some embodiments, provided herein is a method for potent delivery to a cell of a subject comprising administrating to the subject the pharmaceutical composition as described in the present application. In some embodiments of the method, the pharmaceutical composition comprises a pharmaceutical or therapeutic agent assembled with a lipid composition as described in the present application, wherein the lipid composition comprises an ionizable lipid. The lipid composition may further comprise a steroid, a helper lipid, and / or a polymer conjugated lipid.

[0390] In some embodiments of any method described herein, the method of delivery of a therapeutic agent to a target organ (e.g., liver, spleen, or lymph nodes) or a target cell (e.g., a immune cell provided herein; e.g., a T cell) of a subject comprising administering a composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the target organ or target cell that is at least 1.1 -fold greater than a corresponding amount or activity of the therapeutic agent achieved in a non-target organ or non-target cell of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the target organ or target cell is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater4at least 30-fold greater4at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at least 300-fold greater, than a corresponding amount or activity of the therapeutic agent achieved in non-target organ or non- target cell of the subject.

[0391] In some embodiments, the methods of delivery comprise administering a lipid composition described herein provides an effective amount or activity of a therapeutic agent at least 1.1-fold greater than a corresponding amount or activity of the therapeutic agent achieved by administering other compositions. In some embodiments, the effective amount or activity of the therapeutic agent results from administering a lipid composition described herein is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater4at least 30-fold greater4at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at least 300-fold greater, than a corresponding amount or activity of the therapeutic agent achieved by administering other compositions.

[0392] In some embodiments, the methods of delivery comprise administering a lipid described herein provides an effective amount or activity of a therapeutic agent at least 1.1- fold greater than a corresponding amount or activity of the therapeutic agent achieved by administering other lipids. In some embodiments, the effective amount or activity of the therapeutic agent results from administering a lipid described herein is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater at least 30-fold greater at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at least 300-fold greater, than a corresponding amount or activity of the therapeutic agent achieved by administering other lipids.

[0393] In some embodiments, the delivery of the pharmaceutical agent to a cell may alter the genome, transcriptome, or expression levels. The cell may be allowed to, or able to, propagate and the alteration may be passed on to the cells generated from the cell that the therapeutic was delivered to. In this manner, the therapeutic effect may be propagated to a larger number of cells. The alteration to the genome, transcriptome or expression level may also persist in a given cell.

[0394] Also provided herein are methods of treating a disease or disorder using the compounds or the lipid nanoparticle (LNP) compositions described herein. In some embodiments, the methods comprise using the LNPs to deliver a therapeutic pay load for immunotherapy. In some embodiments, the methods comprise using the LNPs to deliver a therapeutic payload to treat a cancer.

[0395] In some embodiments, the compounds or lipid compositions (or pharmaceutical compositions) of the present application can be administrated through any suitable routes comprising parenteral delivery (e.g., injections), such as intravenous, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

[0396] In some embodiments, the compounds or lipid compositions can be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted.

[0397] In some embodiments, the compounds or lipid compositions can be injected into the site of injury, disease manifestation, or pain, for example. In some embodiments, the compounds or lipid compositions can be provided in lozenges for oral, tracheal, or esophageal application. In some embodiments, the compounds or lipid compositions can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines. In some embodiments, the compounds or lipid compositions can be supplied in suppository form for rectal or vaginal application. In some embodiments, the compounds or lipid compositions can even be delivered to the eye by use of creams, drops, or even injection.

[0398] In some embodiments, the delivery of the therapeutic payload or pharmaceutical agent to a cell may alter the genome, transcriptome, or expression levels. The cell may be allowed to, or able to, propagate and the alteration may be passed on to the cells generated from the cell that the therapeutic was delivered to. In this manner, the therapeutic effect may be propagated to a larger number of cells. The alteration to the genome, transcriptome or expression level may also persist in a given cell.

[0399] Dosing Level

[0400] In another aspect, provided is high-potency dosage form of a pharmaceutical agent (or a therapeutic agent) formulated with an ionizable lipid, the dosage form comprising a therapeutic agent (e.g., mRNA encoding a peptide for immunotherapy) assembled with a lipid composition as described herein.

[0401] In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, .005, 0.002, or 0.001 milligram per kilogram (mg / kg, or mpk) body weight, or of a range between (inclusive) any two of the foregoing values.

[0402] In some embodiments, the therapeutic agent is present in the dosage form at a dose of no more than about 10 milligram per kilogram (mg / kg, or mpk) body weight. In some embodiments, the therapeutic agent is present in the dosage form at a dose of no more than about 9 mg / kg, no more than about 8 mg / kg , no more than about 7 mg / kg, no more than about 6 mg / kg, no more than about 5 mg / kg, no more than about 4 mg / kg, no more than about 3 mg / kg, no more than about 2 mg / kg, no more than about 1 mg / kg, no more than about 0.5 mg / kg, no more than about 0.2 mg / kg, no more than about 0.1 mg / kg, no more than about 0.05 mg / kg, or no more than about 0.01 mg / kg. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 5 milligram per milliliter (mg / mL).

[0403] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligram per milliliter (mg / mL), or of a range between (inclusive) any two of the foregoing values.

[0404] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 5 milligram per milliliter (mg / mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 2 milligram per milliliter (mg / mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 1 milligram per milliliter (mg / mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 0.5 milligram per milliliter (mg / mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 0.1 milligram per milliliter (mg / mL).

[0405] In some embodiments, the therapeutic agent (e.g., proteins, nucleic acids) is present in the dosage form at a concentration of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 microgram per milliliter (pg / mL), or of a range between (inclusive) any two of the foregoing values. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1, no more than about 0.5, no more than about 0.2, no more than about 0.1 microgram per milliliter (pg / mL).

[0406] Any suitable dosage form can be prepared for delivery, for example, via oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0407] In some embodiments, the pharmaceutical agent is administered at a dosage of no more than about 10 milligram per kilogram (mg / kg, or mpk) body weight, no more than about 9 mg / kg, no more than about 8 mg / kg , no more than about 7 mg / kg, no more than about 6 mg / kg, no more than about 5 mg / kg, no more than about 4 mg / kg, no more than about 3 mg / kg, no more than about 2 mg / kg, no more than about 1 mg / kg, no more than about 0.5 mg / kg, no more than about 0.2 mg / kg, no more than about 0.1 mg / kg, no more than about 0.05 mg / kg, or no more than about 0.01 mg / kg body weight. In some embodiments, the pharmaceutical agent is administered at a dosage from about 1 pg / kg body weight to about 3 mg / kg body weight.

[0408] In some embodiments, the administration of a dose of the lipid composition provided here can be repeated. If desired, the effective dose of the active lipid composition can be administered as one, two, three, four, five, six or more doses administered separately at appropriate intervals throughout the course of treatment. In some embodiments, the lipid composition can be administered two or three times daily. In some embodiments, the lipid composition will be administered once daily. In some embodiments, the lipid composition is administered about every 1 week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, about every 16 weeks, about every 17 weeks, or about every 18 weeks. In some embodiments, the lipid composition is administered about every 1 month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every 12 months, about every 13 months, about every 14 months, about every 15 months, about every 16 months, about every 17 months, about every 18 months, about every 2 years, about every 2.5 years, about every 3 years, about every 3.5 years, about every 4 years, about every 4.5 years, or about every 5 years. Any subject in need thereof can be treated with the method of the present application. In some embodiments, the subject has been determined to likely respond to the therapeutic agent. For example, the subject may have, is suffering from, or suspected of having a disease or condition. The therapeutic or prophylactic agent(s) as described elsewhere herein may be effective for providing a therapeutic effect for the subject by a variety of mechanisms, for example, via gene therapy (e.g., requiring repeated administration), altered protein production, (e.g., in vivo) chimeric antigen receptor (CAR) T- cell generation, immuno-oncology, vaccine-based approach, reactivation of tumor suppressors, or other mechanisms.

[0409] In some embodiments, the subject has been determined to have a (e.g., missense or nonsense) mutation in a target gene. In some embodiments, the mutation in the target gene is associated with a genetic disease or disorder.

[0410] In some embodiments, the subject has been determined to exhibit an aberrant expression or activity of a protein or polynucleotide that corresponds to a target gene. In some embodiments, the aberrant expression or activity of the protein or polynucleotide is associated with a genetic disease or disorder

[0411] In some embodiments, the subject is selected from the group consisting of mouse, rat, monkey, and human. In some embodiments, the subject is a human.

[0412] In some embodiments, provided herein is a method for targeted delivery of a therapeutic agent to a cell type comprising contacting the cell with the pharmaceutical composition of the present application. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent assembled with a lipid composition as described in the present application, e.g., wherein the lipid composition comprises any of the head or tail groups disclosed herein. In some embodiments, provided herein is a method for potent delivery to a cell of a subject comprising administrating to the subject the pharmaceutical composition as described in the present application. In some embodiments, the pharmaceutical composition comprises a therapeutic payload (e.g., pharmaceutical or therapeutic agent) assembled with a lipid composition as described in the present application, wherein the lipid composition comprises an ionizable lipid. The lipid composition may further comprise a steroid, a helper lipid, and / or a polymer conjugated lipid.

[0413] In some aspects, the methods provided herein can achieve targeted delivery of the therapeutic payload / agent to a target organ or cell. In some embodiments, the target organ comprises the liver, lung, brain, heart, kidneys, or secondary lymphoid tissues (e.g., spleen or lymph nodes). In some embodiments, the target organ comprises the liver. In some embodiments, the target organ comprises the secondary lymphoid tissues. In some embodiments, the target organ comprises the spleen. Target cells provided herein may comprise a cell of the target organ. In some embodiments, the target cell comprises a cell of the liver. A cell of the liver may comprise a hepatocyte, a Kupffer cell, a Stellate cell, a sinusoidal endothelial cell, or a cholangiocyte. In some embodiments, the target cell comprises a neuron or a glial cell. In some embodiments, the target cell comprises an immune cell. In some embodiments, the target cell comprises a lymphocyte (e.g., T cell or B cell). In some embodiments, the target cell comprises an antigen presenting cell (APC). In some embodiments, the APC comprises a macrophage or a dendritic cell.

[0414] In some embodiments, the method comprises delivering a therapeutic payload / agent to a target organ or cell of a subject, thereby providing an effective amount or activity of the therapeutic payload / agent in the target organ or cell that is at least 1.1 -fold greater than the amount or activity achieved in a non-target organ or cell of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the target organ or target cell is at least 1.1-fold greater, at least 1.2-fold greater, at least 1.3-fold greater, at least 1.4-fold greater, at least 1.5-fold greater, at least 1.6-fold greater, at least 1.7-fold greater, at least 1.8-fold greater, at least 1.9-fold greater, at least 2-fold greater, at least 2.1 -fold greater, at least 2.2-fold greater, at least 2.3-fold greater, at least 2.4-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.2-fold greater, at least 3.5-fold greater, at least 3.7-fold greater, at least 4-fold greater, at least 4.2-fold greater, at least 4.5-fold greater, at least 4.7-fold greater, at least 5-fold greater, at least 5.2-fold greater, at least 5.5-fold greater, at least 5.7-fold greater, at least 6- fold greater, at least 6.2-fold greater, at least 6.5-fold greater, at least 6.7-fold greater, at least 7-fold greater, at least 7.5-fold greater, at least 8-fold greater, at least 8.5-fold greater, at least 9-fold greater, at least 9.5-fold greater, at least 10-fold greater, at least 10.5-fold greater, at least 11-fold greater, at least 11.5-fold greater, at least 12-fold greater, at least 12.5-fold greater, at least 13-fold greater, at least 13.5-fold greater, at least 14-fold greater, at least 14.5-fold greater, at least 15-fold greater, at least 16-fold greater, at least 17-fold greater, at least 18-fold greater, at least 19-fold greater, at least 20-fold greater4at least 25-fold greater, at least 30-fold greater^ at least 35-fold greater, at least 40-fold greater, at least 45-fold greater, at least 50-fold greater, at least 55-fold greater, at least 60-fold greater, at least 65-fold greater, at least 70-fold greater, at least 75-fold greater, at least 80-fold greater, at least 85-fold greater, at least 90-fold greater, at least 95-fold greater, at least 100-fold greater, at least 150-fold greater, at least 200- fold greater, at least 250-fold greater, or at least 300-fold greater, than the amount or activity achieved in a non-target organ or cell of the subject.

[0415] In some embodiments, the methods comprise administering the lipid compositions provided herein, thereby providing an effective amount or activity of the therapeutic agent at least 1.1-fold greater than the amount or activity achieved by administering other compositions. In some embodiments, the effective amount or activity of the therapeutic agent results from administering a lipid composition described herein is at least 1.1 -fold greater, at least 1.2-fold greater, at least 1.3-fold greater, at least 1.4-fold greater, at least 1.5-fold greater, at least 1.6- fold greater, at least 1.7-fold greater, at least 1.8-fold greater, at least 1.9-fold greater, at least 2-fold greater, at least 2.1-fold greater, at least 2.2-fold greater, at least 2.3-fold greater, at least

[0416] 2.4-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.2-fold greater, at least

[0417] 3.5-fold greater, at least 3.7-fold greater, at least 4-fold greater, at least 4.2-fold greater, at least

[0418] 4.5-fold greater, at least 4.7-fold greater, at least 5-fold greater, at least 5.2-fold greater, at least 5.5-fold greater, at least 5.7-fold greater, at least 6-fold greater, at least 6.2-fold greater, at least 6.5-fold greater, at least 6.7-fold greater, at least 7-fold greater, at least 7.5-fold greater, at least 8-fold greater, at least 8.5-fold greater, at least 9-fold greater, at least 9.5-fold greater, at least 10-fold greater, at least 10.5-fold greater, at least 11-fold greater, at least 11.5-fold greater, at least 12-fold greater, at least 12.5-fold greater, at least 13-fold greater, at least 13.5- fold greater, at least 14-fold greater, at least 14.5-fold greater, at least 15-fold greater, at least 16-fold greater, at least 17-fold greater, at least 18 -fold greater, at least 19-fold greater, at least 20-fold greater4at least 25-fold greater, at least 30-fold greater4at least 35-fold greater, at least 40-fold greater, at least 45-fold greater, at least 50-fold greater, at least 55-fold greater, at least 60-fold greater, at least 65-fold greater, at least 70-fold greater, at least 75-fold greater, at least 80-fold greater, at least 85-fold greater, at least 90-fold greater, at least 95-fold greater, at least 100-fold greater, at least 150-fold greater, at least 200-fold greater, at least 250-fold greater, or at least 300-fold greater, than the amount or activity achieved by administering other compositions.

[0419] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting comprises administering to a subject the composition comprising the therapeutic agent assembled with the lipid composition.

[0420] Pharmaceutical compositions

[0421] The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. The pharmaceutical composition described herein may comprise a therapeutic or prophylactic composition, or any combination thereof. In some embodiments, the lipidoid compositions can be assembled with mRNA encoding a therapeutic peptide (e.g., antigen-binding peptide or immunomodulator) for immunotherapy. In some embodiments, the individual is a mammal (e.g., a human or a non-human mammal). When administered to an animal (e.g., a human or a non-human animal)the composition or the lipidoid composition is preferably administered as a pharmaceutical composition comprising, for example, a lipidoid composition of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as intravenous or intramuscular injection, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0422] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a lipidoid composition such as a lipidoid composition of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a lipidoid composition of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0423] The phrase "pharmaceutically acceptable" is employed herein to refer to those lipidoid compositions, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0424] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0425] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The lipidoid composition may also be formulated for inhalation. In some embodiments, a lipidoid composition may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0426] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the lipidoid composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0427] Methods of preparing these formulations or compositions include the step of bringing into association an active composition, such as a lipidoid (e.g., nanoparticle) composition as described herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a lipidoid (e.g., nanoparticle) composition as described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0428] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In some embodiments, the pharmaceutical compositions comprising the mRNA / LNPs provided herein are administered through parenteral routes (e.g., intravenous injection or intramuscular injection). Pharmaceutical compositions suitable for parenteral administration comprise one or more active lipidoid compositions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0429] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0430] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0431] Injectable depot forms are made by forming microencapsulated matrices of the subject lipidoid compositions in biodegradable polymers such as poly lactide-poly glycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0432] For use in the methods of this invention, active lipidoid compositions can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0433] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a lipidoid composition at a particular target site.

[0434] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0435] The selected dosage level will depend upon a variety of factors including the activity of the particular lipidoid composition or combination of lipidoid compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular lipidoid composition(s) being employed, the duration of the treatment, other drugs, lipidoid compositions and / or materials used in combination with the particular lipidoid composition(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0436] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or lipidoid composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a lipidoid composition that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the lipidoid composition will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the lipidoid composition, and, if desired, another type of therapeutic agent being administered with the lipidoid composition of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0437] In general, a suitable daily dose of an active lipidoid composition used in the compositions and methods of the invention will be that amount of the lipidoid composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0438] If desired, the effective dose of the active lipidoid composition may be administered as one, two, three, four, five, six or more doses administered separately at appropriate intervals throughout the course of treatment, optionally, in unit dosage forms. In some embodiments of the present invention, the active lipidoid composition may be administered two or three times daily. In some embodiments, the active lipidoid composition will be administered once daily.

[0439] The patient or subject receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0440] In some embodiments, lipidoid compositions of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0441] The present disclosure includes the use of pharmaceutically acceptable salts of lipidoid compositions of the invention in the compositions and methods of the present invention. In some embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra- alkyl ammonium salts. In some embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In some embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In some embodiments, contemplated salts of the invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4- acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d glucoheptonic acid, d gluconic acid, d glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene- 1,5 -disulfonic acid, naphthalene-2- sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1 tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoro acetic acid, and undecylenic acid salts.

[0442] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0443] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0444] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Kits

[0445] Another aspect provides a kit comprising the pharmaceutical composition and / or lipid nanoparticle (LNP) formulations described herein. In some embodiments, the kit can further comprise an apparatus for administering the compositions provided herein, such as appropriate syringes for intravenous or intramuscular administration.

[0446] In any herein-disclosed method, the method further comprises providing instructions for use (IFU), the IFU including instructions for administering the LNP compositions to a subject. In some embodiments, the user instruction directs a user to inject the LNP compositions comprising mRNA intravenously or intramuscularly for use in immunotherapy.

[0447] EXAMPLES

[0448] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0449] Example 1: Chemical Synthesis

[0450] Lipid Synthesis

[0451] Lipids synthesized are listed in TABLE 2. Homogeneous lipids (e.g., lipids comprising one kind of lipid tails) were synthesized through a solvent free Michael Addition reaction between an amine head group and an alkyl-acrylate lipid tail. A headgroup comprising an amine, for example, the aliphatic headgroup 3,3’-Diamino-N-methyldipropylamine, and an acrylate tail were mixed at 1 to 5 molar ratio in Teflon-lined glass screw-top vials at 70 °C for 48 h. The crude products were purified using a Teledyne Isco Chromatography system using methanol / DCM as mobile phase. The purified lipidoids were characterized by electrospray ionization mass spectrometry (ESI-MS).

[0452] In the cases of asymmetric heterogenous lipids (e.g., lipids comprising different lipid tails), the headgroup comprising an amine was mixed with a first kind of lipid tail at 1 to 3.5 molar ratio. After 48 hours of stirring at 70 °C, the partially assembled lipid products were purified as described above. Subsequently the partially assembled lipid products were mixed with a second lipid tail at 1: 1.5 molar ratio. The final asymmetric heterogenous lipids were isolated and verified by ESI-MS.

[0453] In the cases of symmetric heterogenous lipids (e.g., lipids comprising equal numbers of different lipid tails), headgroups comprising Boc-protected amines were reacted with a first kind of lipid tail at 1 to 2.5 molar ratio. After mixing at 70 °C for 48 hours, the half-assembled lipid products were purified. The Boc protecting group was removed by trifluoroacetic acid in DCM, and the half-assembled lipids were further reacted with a second lipid tail to afford the full heterogenous but symmetric lipid products.

[0454] Lipid tail synthesis

[0455] The synthesis of lipid tail O12B is illustrated in FIG. 1A as an example for lipid tail synthesis. Briefly, 2,2’ -dipyridyl disulfide was dissolved in dichloromethane under inert gas in presence of trace amount of acetic acid. 1 -octanethiol solution in dichloromethane was then added dropwise. The reaction mixture was stirred overnight and concentrated by rotary evaporation. Crude reaction mixture was filtered through a short pad of silica gel and flushed with ethyl acetate / hexanes mixture. The collected solvents were concentrated, and the residue was purified by flash column chromatography on silica to obtain Compound 1 in FIG. 1A with good yield. Compound 2 was prepared and purified in a similar way. 2-mercaptoethanol was used as nucleophile for the synthesis of Compound 2 under inert gas. For lipid tail O12B synthesis, Compound 2 was dissolved in dichloromethane under an inert atmosphere and cooled to 0 °C. 1.5 equivalent of triethylamine was added while stirring. Then neat acryloyl chloride (1.2 eq.) was added slowly. Crude reaction mixture was concentrated by rotary evaporation, filtered through a short pad of silica gel and flushed with ethyl acetate / hexanes mixture. The collected solvents were concentrated, and the residue was purified by flash column chromatography on silica to obtain lipid tail with satisfactory overall yield.

[0456] The synthesis of alkene lipid tail replies on a similar scheme to the above-mentioned, wherein the biodegradable alcohol Compound 2 with disulfide bond is replaced with the commercially available unsaturated alcohol cis-5-octene-l-ol.

[0457] The synthesis of Compound 4, a lipid tail comprising carbonate, is illustrated in FIG. IB. Briefly, 4-Nitrophenyl chloroformate (20.2.6 g, 110 mmol) was added in portions to a solution of 2-butyl-l -octanol (18.6 g, 100 mmol) and triethylamine (27.9 ml, 200 mmol) in THF under Argon in a round flask with a magnetic stir bar at room temperature. The reaction mixture was stirred for 2-3 hours until completion, as monitored by TLC. Then the reaction mixture was diluted with ethyl acetate, and washed sequentially with IN HC1 solution, saturated NaHCOa solution, and brine. The organic layers were dried over MgSCL, filtered, and concentrated. The residue was purified by flash column chromatography on silica to afford Compound 3 (31.0 g, 88.3 mmol, 88%).

[0458] Compound 3 (5.62 g, 16 mmol) was dissolved in DMF and added to a solution of 2- hydroxyethyl acrylate (1.86 g, 16 mmol) in DMF in a two-neck round-bottom flask charged with a magnetic stir bar under Ar. The reaction mixture was heated to 50 ° C, and K2CO3 (5.5 g, 40 mmol) was added portion wise to the reaction mixture after 10 min. Then the reaction mixture was heated to 800C for 2-3 hours until completion. DMF was removed by rotary evaporation and the residue was partitioned by addition of water and ethyl acetate. The aqueous layer was extracted with ethyl acetate three times. The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography on silica to afford Compound 4 (3.68 g, 11.2 mmol, 70%). Example 2: LNP Formulation and Characterization

[0459] RNA working solutions (0.134 mg / ml) were prepared in 25 mM sodium acetate buffer using DNase / RNase-free distilled water. Lipid stock solutions (10 mg / mL) were freshly prepared with active lipids or helper lipids (e.g., cholesterol, DOPC or DSPC, or ALC-0159) in ethanol. Reagents were mixed by vortexing and sonicating until the lipids were completely dissolved. A lipid mixture was first prepared by combining the active lipids (e.g., ionizable lipids of the present disclosure) and helper lipids into one vial and mixing thoroughly. Lipid nanoparticles were then formed by mixing the RNA working solution (0.134 mg / ml) and the lipid mixture at 3:1 volume ratio, either manually (for smaller scale) or by Precision Igniter microfluidic device. The formulation solution was transferred into a 300kDa MWCO Float- A- Lyzer G2 Dialysis Device and dialyzed against at least 1000-folds volume of 50 mM Tris buffer overnight at 4 °C. The LNP solution was sterilized with a 0.22 pm filter. TABLE 5 demonstrates an example of a lipid nanoparticle formulation.

[0460] TABLE 5. Example calculation for one LNP formulation

[0461] For LNP characterization, size and polydispersity index (PDI) were determined by dynamic light scattering using a Zetasizer Nano ZS. 10 pL of LNP suspension was transferred to a low-volume cuvette containing 900 pL of Ca2+and Mg2+free PBS at pH 7.4. Refractive index was set to 1.14 and temperature was set to 25 °C. Encapsulation efficiency (EE%) was measured using a fluorescence plate-based assay employing the Ribogreen reagent (Invitrogen) as per PNI Ribogreen assay protocol 16. This assay measures the quantity of mRNA in samples with intact LNPs to determine the quantity of unencapsulated RNA as well as in LNP samples disrupted by triton X-100 to measure the total RNA. EE% is calculated as the difference between the total RNA and the unencapsulated RNA divided by the total RNA. Formulations and results from characterization assays are listed in TABLE 6.

[0462]

[0463] Example 3: mRNA / LNP Delivery to Mice

[0464] IV injection of Luc mRNA / LNP resulted in protein expression in vivo

[0465] All animal experiments were conducted in accordance with the approved animal protocols. LNPs were formulated with lipids and mRNA encoding Luciferase (Luc) based on TABLE 5. LNPs comprising 5 pg mRNA and 50 pg active lipids (e.g., ionizable lipids) were injected intravenously (i.v.) to female adult BALB / c mice (6-8 wk. old). Six hours post injection, 100 pL of luciferin K salt (15 mg / mL in PBS) was administered to the mice through intraperitoneal injections. The mice were then imaged using the In Vivo Imaging System (IVIS) and the total flux of areas of interest was quantified. FIGs. 2A-2G illustrate representative IVIS data using LNPs formulated with active lipids from Sections 1-7 in TABLE 6. As depicted in FIGs. 2A-2G, i.v. injections of Luc mRNA / LNPs to the mice resulted in in vivo production of Luc, primarily observed in the liver.

[0466] IM injection of hEPO mRNA / LNP resulted in protein expression in vivo

[0467] CD-I mice were administered a single intramuscular (IM) dose of 0.004 mg / kg LNP comprising modified mRNA encoded human erythropoietin (hEPO). Blood was collected from mice 6 hours post injection via the submandibular vein. Samples were centrifuged at 3000 xg for 10 minutes and the plasma samples were aliquoted and stored at -80°C until analysis. hEPO concentration in the plasma was determined via ELISA, and quantification analysis was performed using Softmax Pro (Molecular device). Results were shown in FIGs. 3A-3C as mean ± SD. As depicted in FIGs. 3A-3C, hEPO was detected in all samples tested, indicating that the LNP formulations used successfully delivered the modified hEPO mRNA to inside the cells.

[0468] Example 4: In Vivo Testing of Organ- Targeting Lipids

[0469] To identify lipid formulations that preferentially deliver mRNA to secondary lymphoid tissues, we tested 76 lipid formulations in three different experiments (30+30+16). In each experiment, formulation with different ionizable lipids encapsulating luciferase mRNA was injected into CD1 mice through tail- vein injection. After 5 hours delivery and transfection, luciferin substrate was injected to the animals and the animals were imaged by IVIS imager. Subsequently the animal was sacrificed and dissected. Spleen tissue as well as liver tissue were isolated and imaged ex vivo. Ex vivo luciferase total flux in the spleen tissue were compared to that in the whole animal or in the liver tissue. We identified lipids and their formulations that preferentially deliver the Luc mRNA to the spleen, as shown in TABLE 7. Exemplary images from IVIS are depicted in FIGs. 8A-8B, showing strong spleen-targeting delivery.

[0470] TABLE 7. Luciferase flux of spleen-targeting LNPs

[0471] Additionally, we analyzed and characterized the cell type specificity of the spleentargeting lipids. Formulation with the spleen-targeting lipids encapsulating mCre mRNA were injected into Ail4 transgenic mice intravenously, and after 7 days the mice were sacrificed and dissected. The tissues (e.g., spleen) were extracted, homogenized, and subjected to flow cytometry to identify the TdTomato positive cells. The percentage of TdTomato positive cells were calculated in different cell types, as shown in FIG. 9. Antigen presenting cells (APC) such as dendritic cells and macrophage in the spleen showed a higher transfection rate.

[0472] We also identified lipid formulations that preferentially target the liver. As shown in TABLE 8, the lipids and formulations showed strong luciferase signal (>10nROI reading), surpassing current commercially available liver delivering lipids. Exemplary images from IVIS are depicted in FIG. 10, showing strong liver- targeting delivery.

[0473] TABLE 8. Luciferase flux of liver-targeting LNPs

Claims

We claim:

1. A composition, comprising a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid has an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I):or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine headgroup;Ri and R2 are each independently a C1-C12 bivalent aliphatic or heteroaliphatic radical;X isindependently, a bond, O, S, or NRC; G is O, S, or NRd; Q is ORe, SRf, or NRgRh; and each of r and t is independently 1-6; each of Rc, Rd, Re, Rf, Rg, and Rhis independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl;Y and U are each independently a bond, O, S, NR10, or Se; n is 0 or 1 ;R3 and R4, are each independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atom to which they are attached, form C=O;R5is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for immunotherapy .

2. The composition of claim 1, wherein the amine head group is represented by:wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, Cl -20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl,C1-C20 bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.

3. The composition of claim 1 or 2, wherein the ionizable lipid is represented by Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Rbis a substituted or unsubstituted alkyl; nl and n2 are each independently 1, 2, 3, 4, 5, or 6; andRbl, Rb2, Rb3and Rb4are each independentlywherein at least one of Rbl, Rb2, Rb3and Rb4is not H.

5. The composition of any one of claims 1-4, wherein the at least one hydrophobic tailwherein Rki and Rk3 are each independently a C1-C10 alkyl;Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, Cl- C20 heterocycloalkyl, aryl, or heteroaryl;Rk4 and Rks are each independently H, or C1-C10 alkyl.

6. The composition of any one of claims 1-5, wherein the lipid composition further comprises a steroid.

7. The composition of claim 6, wherein the steroid is cholesterol or a cholesterol derivative.

8. The composition of any one of claims 1-7, wherein the lipid composition furthercomprises a helper lipid.

9. The composition of claim 8, wherein the helper lipid is l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

10. The composition of any one of claims 1-9, wherein the lipid composition further comprises a polymer conjugated lipid.

11. The composition of claim 10, wherein the polymer conjugated lipid is a PEG conjugated lipid.

12. The composition of claim 11, wherein the polymer conjugated lipid is 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2k).

13. The composition of claim 1, wherein the lipid composition comprises a transactivator of transcription (TAT) peptide modification.

14. The composition of any one of claims 1-13, wherein the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid.

15. The composition of any one of claims 1-14, wherein the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.

16. The composition of any one of claims 6-15, wherein the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%.

17. The composition of any one of claims 8-16, wherein the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.

18. The composition of any one of claims 10-17, wherein the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.

19. The composition of any one of claims 14-18, wherein the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16 / 4 / 1 / 1.

20. The composition of any one of claims 14-18, wherein the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.8 / 4 / 2 / 1.

21. The composition of any one of claims 14-18, wherein the weight ratio of the pharmaceutical agent / lipid composition is from about 1:200 to about 1:5.

22. The composition of any one of claims 1-6, wherein the lipid composition further comprises a steroid and a helper lipid.

23. The composition of claim 22, wherein the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.

24. The composition of claim 22 or 23, wherein the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%.

25. The composition of any one of claims 22-24, wherein the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%.

26. The composition of any one of claims 22-25, wherein the weight ratio of the ionizable lipid / steroid / helper lipid is about 2 / 1 / 1.

27. The composition of any one of claims 1-26, wherein the lipid composition further comprises an excipient.

28. The composition of claim 27, wherein the excipient is selected from the group consisting of (2-hydroxypropyl)-P-cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundec anoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, 80-EC16, and Bile salts.

29. The composition of claim 27, wherein the excipient is present in the composition at a weight percentage from about 5% to about 60%.

30. The composition of any one of claims 1-29, wherein the pharmaceutical agent further comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof.

31. The composition of any one of claims 1-29, wherein the pharmaceutical agent comprises:(a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or(b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof.

32. The composition of claim 31, wherein the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid.

33. The composition of claim 31 or 32, wherein the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease.

34. The composition of any one of claims 1-33, wherein the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical.

35. The composition of any one of claims 1-33, wherein the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical.

36. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering an effective amount of the composition according to any one of claims 1-35.

37. The method of claim 36, wherein the target organ comprises a liver, a spleen or a lymph node.

38. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according to any one of claims 1-35 that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organin the subject as compared to that achieved absent the lipid composition.

39. The method of claim 38, wherein the target organ comprises a liver, a spleen or a lymph node.

40. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according to any one of claims 1-35 that comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to a non-target organ.

41. The method of claim 40, wherein the target organ comprises a liver, a spleen or a lymph node.

42. The method of any one of claims 36-41, wherein the administering is through systemic administration.

43. The method of any one of claims 36-41, wherein the administering is through intramuscular administration.

44. The method of any one of claims 36-41, wherein the administering is through intravenous administration.

45. The method of any one of claims 36-44, wherein the pharmaceutical agent is delivered at a dosage of no more than 3 mg / kg body weight.

46. The method of claim 45, wherein the pharmaceutical agent is delivered at a dosage from about 1 pg / kg body weight to about 3 mg / kg body weight.

47. The method of any one of claims 36-46, wherein the pharmaceutical agent is delivered in one or more doses.

48. The composition of any one of claims 1-35, wherein the hydrophobic tail comprises a structure selected from Table 1.

49. The composition of any one of claims 1-35, wherein the ionizable lipid has a structure selected from Table 2.

50. A compound represented by Formula (III):Head - (RLipid)p (Formula III) or a pharmaceutically acceptable salt thereof; wherein:wherein each Ra is independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each RL1Pldis independently selected from:each Rkl and Rk3 are independently selected from C1-C10 alkyl; each Rk2 is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4 and Rk5 are independently selected from H and C1-C10 alkyl; s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6.

51. The compound or pharmaceutically acceptable salt of claim 50, wherein RLipidis52. The compound or pharmaceutically acceptable salt of claim 50, wherein Head is selected from the group consisting of53. The compound or pharmaceutically acceptable salt of claim 50, wherein RL1Pldis represented by a structure in Table 1 or Table 3.

54. The compound or pharmaceutically acceptable salt of claim 50, wherein the compound is represented by a structure in Table 2 or Table 4.

55. A composition, comprising a pharmaceutical agent assembled with a lipid composition comprising an ionizable lipid represented by Formula (III):Head - (RLipid)p (Formula III) or a pharmaceutically acceptable salt thereof; wherein:wherein each Ra is independently selected from H, C1-C20 alkyl, C2-C20 alkenyl, C2- C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20- membered heteroalkyl, C3-C7 carbocycle, and 3- to 7-membered heterocycle; each Rb is independently selected from H and C1-C5 alkyl; each RL1Pldis independently selected from:each Rkl and Rk3 are independently selected from C1-C10 alkyl; each Rk2 is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; each Rk4 and Rk5 are independently selected from H and C1-C10 alkyl; s is selected from 0, 1, and 2; each q is selected from 1, 2, 3, and 4; each w is selected from 1, 2, 3, and 4; and p is selected from 1, 2, 3, 4, 5, and 6.

56. A composition comprising a pharmaceutical agent assembled with a lipid composition comprising an ionizable lipid, wherein the ionizable lipid has an amine head group and at least one hydrophobic tail RLipidhaving a structure of Formula (I):or a pharmaceutically acceptable salt thereof; wherein: indicates a point of attachment to a nitrogen in the amine headgroup;R1 and R2 are each independently selected from C1-C12 alkylene, C1-C12 alkenylene, and 1- to 12- membered heteroalkylene;X is each of L3, L4, L5, and L6 is independently selected from a bond, O, S, and NRc;G is selected from O, S, and NRd;Q is selected from ORe, SRf, and NRgRh; r is selected from 1, 2, 3, 4, 5, and 6;t is selected from 1, 2, 3, 4, 5, and 6; each of Rc, Rd, Re, Rf, Rg, and Rh is independently selected from H, C1-C10 alkyl, 1- to 10- membered heteroalkyl, aryl, and heteroaryl;Y and U are each independently selected from a bond, O, S, NR10, and Se; n is 0 or 1 ;R3 and R4, are each independently selected from H, Cl -CIO alkyl, 1- to 10-membered heteroalkyl, aryl, and heteroaryl; or R3 and R4 together with the atom to which they are attached to form =0;R5 is selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl;RIO is selected from H and C1-C5 alkyl; and the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for immunotherapy .

57. The composition of claim 56, wherein the at least one hydrophobic tail RLipidhas awherein: each Rkl and Rk3 is independently selected from C1-C10 alkyl; each Rk2 is independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 1- to 20-membered heteroalkyl, 3- to 20-membered heterocycloalkyl, aryl, and heteroaryl; and each Rk4 and Rk5 is independently selected from H and Cl -CIO alkyl.

58. The composition of claim 56 or claim 57, wherein the amine head group is represented by:wherein Ra, Ra’, Ra”, and Ra’” are each independently selected from H, C1-C20 alkyl, C2- C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, 3- to 20-membered heterocycloalkyl, 1- to 20-membered heteroalkyl, C6-C20 aryl, and 6- to 20- membered heteroaryl; andZ is selected from C1-C20 alkylene, a 1- to 20-membered heteroalkylene, arylene, and heteroarylene.

59. The composition of any one of claims 56-58, wherein the amine headgroup is represented by Formula (II):or a pharmaceutically acceptable salt thereof, wherein:Rb is substituted or unsubstituted C1-C6 alkyl; andnl and n2 are each independently selected from 1, 2, 3, 4, 5, and 6.

61. The composition of any one of claims 55-60, wherein the lipid composition further comprises a steroid.

62. The composition of claim 61, wherein the steroid is cholesterol or a cholesterol derivative.

63. The composition of any one of claims 55-62, wherein the lipid composition further comprises a helper lipid.

64. The composition of claim 63, wherein the helper lipid is l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

65. The composition of any one of claims 55-64, wherein the lipid composition further comprises a polymer conjugated lipid.

66. The composition of claim 65, wherein the polymer conjugated lipid is a PEG conjugated lipid.

67. The composition of claim 66, wherein the polymer conjugated lipid is 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2k).

68. The composition of any one of claims 55-67, wherein the composition comprises a transactivator of transcription (TAT) peptide modification.

69. The composition of any one of claims 55-68, wherein the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid.

70. The composition of any one of claims 55-69, wherein the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.

71. The composition of any one of claims 61-70, wherein the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%.

72. The composition of any one of claims 63-71, wherein the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.

73. The composition of any one of claims 65-72, wherein the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.

74. The composition of any one of claims 69-73, wherein the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16 / 4 / 1 / 1.

75. The composition of any one of claims 69-73, wherein the weight ratio of the ionizable lipid / steroid / helper lipid / polymer conjugated lipid is about 16.6 / 4 / 2 / 1.

76. The composition of any one of claims 55-75, wherein the weight ratio of the pharmaceutical agent / lipid composition is from about 1:200 to about 1:5.

77. The composition of any one of claims 55-60, wherein the lipid composition furthercomprises a steroid and a helper lipid.

78. The composition of any one of claims 55-77, wherein the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.

79. The composition of any one of claims 63-78, wherein the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%.

80. The composition of any one of claims 61-69, wherein the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%.

81. The composition of any one of claims 77-79, wherein the weight ratio of the ionizable lipid / steroid / helper lipid is about 2 / 1 / 1.

82. The composition of any one of claims 55-81, wherein the composition further comprises an excipient.

83. The composition of claim 82, wherein the excipient is selected from the group consisting of (2-hydroxypropyl)-P-cyclodextrin ((HP-P-CD), stearic acid, Perfluoroundec anoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, 80-EC16, and Bile salts.

84. The composition of claim 83, wherein the excipient is present in the composition at a weight percentage from about 5% to about 60%.

85. The composition of any one of claims 55-84, wherein the pharmaceutical agent further comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof.

86. The composition of any one of claims 55-85, wherein the pharmaceutical agent comprises:(a) a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof; or(b) a polynucleotide that encodes a gene modulating moiety configured to specifically bind at least a portion of a target gene or a gene product thereof.

87. The composition of claim 86, wherein the gene modulating moiety comprises a guide nucleic acid configured to complex with at least a portion of the target gene or the gene product thereof, or a polynucleotide sequence that encodes the guide nucleic acid.

88. The composition of claim 86 or 87, wherein the gene modulating moiety comprises a heterologous endonuclease or a polynucleotide comprising a sequence that encodes the heterologous endonuclease.

89. The composition of any one of claims 55-88, wherein the ionizable lipid comprises at least two RLipid, wherein not all of the at least two RLipid are identical.

90. The composition of any one of claims 55-89, wherein the ionizable lipid comprises at least two RLipid, wherein two or more RLipid are identical.

91. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering an effective amount of the composition according to any one of claims 55-90.

92. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according to any one of claims 55-90, wherein the lipid compositioncomprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to that achieved absent the lipid composition.

93. A method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition according to any one of claims 55-90, wherein the lipid composition comprises an ionizable lipid, a steroid, a helper lipid, and a polymer conjugated lipid; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organin the subject as compared to a non-target organ.

94. The method of any one of claims 91-93, wherein the administering is through systemic administration.

95. The method of any one of claims 91-94, wherein the administering is through intramuscular administration.

96. The method of any one of claims 91-94, wherein the administering is through intravenous administration.

97. The method of any one of claims 91-96, wherein the pharmaceutical agent is delivered at a dosage of no more than 3 mg / kg body weight.

98. The method of claim 97, wherein the pharmaceutical agent is delivered at a dosage from about 1 pg / kg body weight to about 3 mg / kg body weight.

99. The method of any one of claims 91-98, wherein the pharmaceutical agent is delivered in one or more doses.

100. The method of any one of claims 91-99, wherein the target organ comprises a liver.

101. The method of any one of claims 91-99, wherein the target organ comprises a spleen or a lymph node.

102. The compound or pharmaceutically acceptable salt of claim 50, wherein RLipidis represented by a structure in Table 3.

103. The compound or pharmaceutically acceptable salt of claim 50, wherein the compound is represented by a structure in Table 4.

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