Lipid Compositions Comprising Peptide-Lipid Conjugates

Abstract

A lipid composition containing a nucleic acid is provided, the lipid composition comprising a peptide-lipid conjugate. The peptide of the peptide-lipid conjugate can be 4 to 52 amino acids in length. A method of using the lipid composition in in vivo delivery of a nucleic acid is further provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 184,584, filed May 5, 2021, which is incorporated by reference in its entirety and for all purposes.

[0002] The present disclosure relates to lipid conjugates. More specifically, the present disclosure relates to peptide-lipid conjugates useful in lipid delivery technologies. [Background technology]

[0003] The delivery of therapeutic agents to cells or tissues of human subjects is important for their therapeutic effect and is usually hindered by the limited ability of the compounds to reach the target cells and tissues. Many macromolecules and molecules with a net ionic charge face multiple barriers when entering cells, and the problem becomes even more complicated when such agents must be delivered to a specific cell type of interest. Unlike small molecule drugs, these types of molecules do not undergo passive diffusion across the cell membrane. Biologically active proteins such as immunoglobulins, as well as potential therapeutic agents of the polynucleotide classes such as genomic DNA, cDNA, mRNA, and siRNA, antisense oligonucleotides, and even certain low molecular weight peptides, peptide hormones, and antibiotics are some of the examples of biologically active molecules for which effective targeting to patient tissues is often not achieved.

[0004] Although some gene therapies can successfully utilize viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and simplicity of large-scale production. One of the most significant advances in lipid-based nucleic acid therapeutics occurred in August 2018, when patisiran (ALN-TTR02) was approved by the U.S. Food and Drug Administration (FDA) and the European Commission (EC) as the first siRNA therapeutic. ALN-TTR02 is an siRNA formulation based on the so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of patisiran, the delivery of nucleic acid therapeutics via lipid formulations is still under development.

[0005] Lipid-based formulations often have polyethylene glycol (PEG)-based compounds as one of the components. PEG can be conjugated with lipids, cholesterol, cationic polymers, or other compounds to facilitate integration into lipid-based formulations. Typically, PEG is included in lipid formulations as a coating or surface ligand, a technique called PEGylation, which prevents lipid particles, liposomes, micelles, etc. from agglomerating, protects lipid-based formulations from the immune system, and helps them escape from reticuloendothelial system (RES) uptake (Nanomedicine (Lond) 2011 Jun;6(4):715-28). PEGylation is widely used to stabilize lipid formulations and their payloads through physical, chemical, and biological mechanisms. Detergent-like PEG lipids (e.g., PEG-DSPE) can enter lipid formulations and form a hydration layer and a steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types: brush-like layer and mushroom-like layer. Increased PEGylation has been shown to lead to a significant increase in the circulating half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15;13:507-30; J. Control Release. 2010 Aug 3;145(3):178-81).

[0006] Despite the advantages and uses of PEG conjugates in lipid-based formulations, the use of PEG is also associated with some problems. For example, a study on intracellular delivery of nucleic acids by Song et al. found that PEG lipids severely inhibited the transport of active nucleic acids and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm (Song, LY, et al. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2002 1558(1):1-13). Furthermore, PEG, as a molecule that does not naturally occur in biological systems, is associated with undesirable immunogenic responses (Garay and Labaune. The Open Conference Proceedings Journal. Vol. 2. No. 1. 2011). After decades of use of PEGylated drugs in human therapeutics, it has been observed that treating patients with PEGylated drugs can lead to the formation of antibodies that specifically recognize and bind to PEG (anti-PEG antibodies). Anti-PEG antibodies were also found in patients who had never been treated with PEGylated drugs but had consumed products containing PEG (Hoang Thi et al. Polymers 12(2):298.2020). Thus, treating patients who produce anti-PEG antibodies with PEGylated drugs can lead to accelerated blood clearance, reduced drug efficacy, hypersensitivity, and potentially life-threatening side effects.

[0007] Several alternative polymers have been investigated as potential replacements for PEGylation in pharmaceutical compositions. Some of these include hydrophilic polymers such as polyoxazolines, poly(N-vinylpyrrolidone), poly(glycerol), and polyacrylamides, natural polymers such as lipids, carbohydrates, and proteins (e.g., serum albumin), as well as investigations of polyamino acids or zwitterionic polymers such as poly(carboxybetaine), poly(sulfobetaine), and phosphobetaine-based polymers (Hoang Thi et al. 2011). Many of these polymers are found in everyday products or other pharmaceutical compositions and run the risk of generating an immunogenic response. One protein that has attracted some interest is the XTEN peptide technology, which is available in peptide sizes of 144, 288, 432, 576, and 864 amino acid residues in length and fused to therapeutic peptides and proteins to increase their in vivo half-life (Podust et al. Journal of Controlled Release 240 (2016): 52-66). Although significant development has been made in finding alternatives to PEGylated compositions, XTEN and other tested polymers are mainly characterized by their ability to increase in vivo half-life and their tendency to be too large for nucleic acid lipid delivery applications.In addition, any PEGylated alternative for nucleic acid lipid delivery must be able to be conjugated with suitable lipids that can achieve not only desirable in vivo half-life, but also target cell uptake and acceptable shedding rate from lipid formulations.Therefore, there is a need for new PEG alternatives that are particularly suitable for the unique needs of nucleic acid lipid delivery compositions. Summary of the Invention

[0008] Additional features and advantages of the subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the written description and embodiments herein, as well as the accompanying drawings.

[0009] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology.

[0010] The present disclosure provides compositions of peptides, peptidomimetics, and their conjugates that can be used in the formulation of lipid formulations that encapsulate drug molecules, including oligonucleotide drugs such as ribonucleic acid and deoxyribonucleic acid. These peptides, peptidomimetics, and their conjugates can show superior performance than PEG conjugates in the delivery of nucleic acid therapeutics in vivo. Although the peptides may contain repeating units of serine, threonine, glutamic acid, and proline as tetrapeptides (STEP peptides), such repeating sequences are not the only arrangements that can achieve the superior performance results of PEG-lipid conjugates. Specifically, the inclusion of a threshold amount of hydrophilic amino acids or derivatives thereof can potentially form a "water cage" around the lipid particle through hydrogen bonding interactions with the amino acid side chains. Furthermore, the inclusion of a threshold amount of proline, an amino acid that imparts rigidity to the structure of the peptide, further provides a preferred conformation of the peptide portion of the peptide-lipid conjugate that can promote the formation of the water cage. This water layer likely acts as a steric barrier to the interaction of LNPs with blood components, preventing opsonization, complement activation, and early clearance while the LNPs are in circulation. Furthermore, this peptide conjugation approach and formulation technique allows the incorporation of a variety of peptides with tunable properties into the LNP matrix, so that their function, cell and tissue specificity, and pharmacokinetic and toxicological properties can be adjusted and optimized to meet the requirements of different applications.

[0011] In one embodiment, a lipid composition containing a nucleic acid is provided, wherein the lipid composition comprises a peptide-lipid conjugate.

[0012] In one embodiment, a lipid composition is provided comprising a peptide-lipid conjugate and a nucleic acid, i. the peptide of the peptide-lipid conjugate consists of about 4 to about 52 amino acids; ii. at least about 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline; iii. about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have hydrophilic side chains; iv. less than about 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine; v. the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol; vi. The peptide-lipid conjugate comprises from about 0.1 mol % to about 10 mol % of all lipids in the lipid composition.

[0013] In some embodiments, methods are provided for delivering a lipid composition of the present disclosure to a target. In some embodiments, methods are provided for treating a disease in a subject, the method comprising administering a lipid composition of the present disclosure.

[0014] Various features of exemplary embodiments of the invention are described below with reference to the drawings. The illustrated embodiments are intended to illustrate, but not to limit, the invention. The drawings include the following figures: [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the effect of using a peptide-lipid conjugate described herein in a lipid nanoparticle formulation compared to PEG on in vivo expression of human erythropoietin (hEPO) expression levels (ng / ml) as described in Example 7. [Diagram 2]FIG. 2 shows the effect of using a peptide-lipid conjugate described herein in a lipid nanoparticle formulation compared to PEG on in vivo knockdown of Factor VII (FVII) normalized to a phosphate buffered saline (PBS) baseline as described in Example 8. [Diagram 3] Figure 3 shows representative images of liver and spleen sections stained for detection of tdTomato protein expression. mRNA enabling tdTomato expression was delivered to the organs by injection of lipid nanoparticle (LNP) formulations containing peptide 7 or DMG-PEG conjugates, as described in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Various configurations of the subject technology will be readily apparent to those skilled in the art from this disclosure, and it will be understood that various configurations of the subject technology have been shown and described by way of example. It will be understood that the subject technology is capable of other configurations and different configurations, and all of its several details can be modified in various other respects without departing from the scope of the subject technology. Accordingly, the Summary of the Invention, Brief Description of the Drawings, and Detailed Description of the Invention should be regarded as illustrative in nature, and not restrictive.

[0017] The detailed description of the invention described below is intended as an illustration of various configurations of the subject technology, and is not intended to represent the only configurations in which the subject technology may be practiced. The accompanying drawings are incorporated in this specification and constitute a part of the detailed description of the invention. The detailed description of the invention includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. Similar components are labeled with the same element numbers for ease of understanding.

[0018] In one embodiment, a lipid composition containing a nucleic acid is provided, wherein the lipid composition comprises a peptide-lipid conjugate.

[0019] In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 12 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 16 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 20 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 24 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 28 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 32 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 36 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 40 to about 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 44 to about 52 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 48 to about 52 amino acids in length.

[0020] In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 48 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 44 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 40 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 36 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 32 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 28 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 24 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 20 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 16 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 12 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 to about 8 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 4 amino acids long, 8 amino acids long, 12 amino acids long, 16 amino acids long, 20 amino acids long, 24 amino acids long, 28 amino acids long, 32 amino acids long, 36 amino acids long, 40 amino acids long, 44 amino acids long, 48 amino acids long, or 52 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 12 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 12 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 16 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 16 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 20 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 20 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 24 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 24 amino acids in length.In some embodiments, the peptide of the peptide-lipid conjugate is about 28 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 28 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 32 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 32 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 36 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 36 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is about 40 amino acids long. In some embodiments, the peptide of the peptide-lipid conjugate is 40 amino acids long.

[0021] In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 50 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 44 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 40 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 36 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 32 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 28 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 24 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 8 to about 20 amino acids in length. In some embodiments, the peptide of the peptide-lipid conjugate is about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, or 52 amino acids in length.

[0022] In some embodiments, at least about 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline. In some embodiments, about 14% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 20% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 25% to about 60% of the amino acids in the peptide are proline.

[0023] In some embodiments, about 14% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 20% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 25% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 30% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 35% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 40% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 45% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 50% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 55% to about 60% of the amino acids in the peptide are proline.

[0024] In some embodiments, about 14% to about 60% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 55% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 50% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 45% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 40% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 35% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 30% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 25% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 20% of the amino acids in the peptide are proline. In some embodiments, about 14% to about 15% of the amino acids in the peptide are proline. In some embodiments, about 14%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the amino acids in the peptide are proline.

[0025] In some embodiments, about 28% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 35% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 40% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 45% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 50% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 55% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 60% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 65% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 70% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 75% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 80% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain.

[0026] In some embodiments, about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 75% to about 85% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 70% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 65% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 60% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 55% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 50% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 45% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 40% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 28% to about 35% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain.

[0027] In some embodiments, about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. In some embodiments, about 35% to about 85% of the amino acids in the peptide have a hydrophilic side chain. In some embodiments, about 35% to about 80% of the amino acids in the peptide have a hydrophilic side chain. In some embodiments, about 40% to about 75% of the amino acids in the peptide have a hydrophilic side chain. In some embodiments, about 45% to about 85% of the amino acids in the peptide have a hydrophilic side chain. In some embodiments, about 50% to about 75% of the amino acids in the peptide have a hydrophilic side chain. In some embodiments, about 28%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain.

[0028] In some embodiments, the amino acids having hydrophilic side chains are independently selected from glutamine, glutamic acid, asparagine, aspartic acid, serine, OC 1-6 Alkylserine, Threonine, and OC 1-6 In some embodiments, the amino acid having a hydrophilic side chain is selected from glutamine, glutamic acid, asparagine, aspartic acid, serine, OC 1-6 Alkylserine, Threonine, and OC 1-6 In some embodiments, the amino acid having a hydrophilic side chain comprises an alkyl threonine. In some embodiments, the amino acid having a hydrophilic side chain comprises glutamine. In some embodiments, the amino acid having a hydrophilic side chain comprises glutamic acid. In some embodiments, the amino acid having a hydrophilic side chain comprises asparagine. In some embodiments, the amino acid having a hydrophilic side chain comprises aspartic acid. In some embodiments, the amino acid having a hydrophilic side chain comprises serine. In some embodiments, the amino acid having a hydrophilic side chain comprises OC 1-6 In some embodiments, the amino acid having a hydrophilic side chain comprises OC alkylserine. In some embodiments, the amino acid having a hydrophilic side chain comprises threonine. In some embodiments, the amino acid having a hydrophilic side chain comprises OC alkylserine. 1-6 Contains alkyl threonine.

[0029] In some embodiments, the OC 1-6 Alkylserine is OC 1 In some embodiments, OC 1-6 Alkylserine is OC 2 In some embodiments, OC 1-6 Alkylserine is OC 3 In some embodiments, OC 1-6 Alkylserine is OC 4 In some embodiments, OC 1-6 Alkylserine is OC 5 In some embodiments, OC 1-6 Alkylserine is OC 6 It is an alkylserine.

[0030] In some embodiments, the OC 1-6 Alkylthreonine is OC 1 In some embodiments, OC 1-6 Alkylthreonine is OC 2 In some embodiments, OC 1-6 Alkylthreonine is OC 3 In some embodiments, OC 1-6 Alkylthreonine is OC 4 In some embodiments, OC 1-6 Alkylthreonine is OC 5 In some embodiments, OC 1-6 Alkylthreonine is OC 6 It is an alkyl threonine.

[0031] In some embodiments, less than about 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine. In some embodiments, less than about 30% of the amino acids in the peptide are glycine. In some embodiments, less than about 20% of the amino acids in the peptide are glycine. In some embodiments, less than about 10% of the amino acids in the peptide are glycine. In some embodiments, less than about 5% of the amino acids in the peptide are glycine. In some embodiments, less than about 4% of the amino acids in the peptide are glycine. In some embodiments, less than about 3% of the amino acids in the peptide are glycine. In some embodiments, less than about 2% of the amino acids in the peptide are glycine. In some embodiments, the peptide has no glycine.

[0032] In some embodiments, the lipid composition is selected from a lipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion. In some embodiments, the lipid composition is a lipoplex. In some embodiments, the lipid composition is a liposome. In some embodiments, the lipid composition is a polymer-based carrier. In some embodiments, the lipid composition is an exosome. In some embodiments, the lipid composition is a lamellar body. In some embodiments, the lipid composition is a micelle. In some embodiments, the lipid composition is an emulsion. In some embodiments, the lipid composition is a lipid nanoparticle.

[0033] In some embodiments, the liposomes are selected from cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In some embodiments, the liposomes are cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In some embodiments, the liposomes are cationic liposomes. In some embodiments, the liposomes are nanoliposomes. In some embodiments, the liposomes are proteoliposomes. In some embodiments, the liposomes are unilamellar liposomes. In some embodiments, the liposomes are multilamellar liposomes. In some embodiments, the liposomes are ceramide-containing nanoliposomes. In some embodiments, the liposomes are multivesicular liposomes.

[0034] In some embodiments, the lipid nanoparticles have a size of less than about 200 nm.

[0035] In some embodiments, the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol.

[0036] In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.4 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.7 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 1 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 1.3 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 1.6 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 1.9 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 2.2 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 2.5 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 2.8 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 3.1 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 3.4 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 3.7 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 4 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 4.3 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 4.6 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 4.9 mol % to about 10 mol % of all lipids in the lipid composition.In some embodiments, the peptide-lipid conjugate comprises about 5.2 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 5.5 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 5.8 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 6.1 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 6.4 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 6.7 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 7 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 7.3 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 7.6 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 7.9 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 8.2 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 8.5 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 8.8 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 9.1 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 9.4 mol% to about 10 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 9.7 mol % to about 10 mol % of all lipids in the lipid composition.

[0037] In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 9.7 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 9.4 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 9.1 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 8.8 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 8.5 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 8.2 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 7.9 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 7.6 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 7.3 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 7 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 6.7 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 6.4 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 6.1 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 5.8 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 5.5 mol% of the total lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 5.2 mol% of the total lipids in the lipid composition.In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 4.9 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 4.6 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 4.3 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 4 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 3.7 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 3.4 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 3.1 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 2.8 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 2.5 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 2.2 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 1.9 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 1.6 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 1.3 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 1 mol% of all lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 0.7 mol% of the total lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol% to about 0.4 mol% of the total lipids in the lipid composition.In some embodiments, the peptide-lipid conjugate comprises about 0.1 mol%, 0.4 mol%, 0.7 mol%, 1 mol%, 1.3 mol%, 1.6 mol%, 1.9 mol%, 2.2 mol%, 2.5 mol%, 2.8 mol%, 3.1 mol%, 3.4 mol%, 3.7 mol%, 4 mol%, 4.3 mol%, 4.6 mol%, 4.9 mol%, 5.2 mol%, 5.5 mol%, 5.8 mol%, 6.1 mol%, 6.4 mol%, 6.7 mol%, 7 mol%, 7.3 mol%, 7.6 mol%, 7.9 mol%, 8.2 mol%, 8.5 mol%, 8.8 mol%, 9.1 mol%, 9.4 mol%, 9.7 mol%, or 10 mol% of the total lipids in the lipid composition.

[0038] In some embodiments, the peptide-lipid conjugate comprises about 0.5 mol% to about 5 mol% of the total lipids in the lipid composition. In some embodiments, the peptide-lipid conjugate comprises about 1.0 mol% to about 3 mol% of the total lipids in the lipid composition.

[0039] In some embodiments, the lipid composition encapsulates the nucleic acid.

[0040] In some embodiments, the lipid composition is complexed with a nucleic acid.

[0041] In some embodiments, the lipid of the peptide-lipid conjugate is conjugated to the peptide via a linker. In some embodiments, the linker is a bond, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. In embodiments, the linker is a substituted or unsubstituted alkyl (e.g., C 1 ~C 8 , C 1 ~C 6 , or C 1 ~C 4), substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl (e.g., 2-8 membered, 2-6 membered, or 2-4 membered), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C 6 -C 10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In some embodiments, the linker is a substituted or unsubstituted alkyl. In some embodiments, the linker is a substituted or unsubstituted heteroalkyl. In some embodiments, the linker is a substituted or unsubstituted cycloalkyl. In some embodiments, the linker is a substituted or unsubstituted heterocycloalkyl. In some embodiments, the linker is a substituted or unsubstituted aryl. In some embodiments, the linker is a substituted or unsubstituted heteroaryl.

[0042] In some embodiments, the linker is -S-, -C(O)O-, amido (-C(O)NH-), or amino (-NR N -), wherein R N , H, C 1-6 Alkyl, carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH 2 CH 2 C(O)-), succinamidyl (-NHC(O)CH 2 CH 2 In some embodiments, the linker is selected from -C(O)NH-), ether, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), and sulfonate ester. In some embodiments, the linker comprises -S-. In some embodiments, the linker comprises -C(O)O-. In some embodiments, the linker has a structure comprising an amide (-C(O)NH-). In some embodiments, the linker is an amino (-NR N -), wherein R N , H, C 1-6Alkyl, carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH 2 CH 2 C(O)-), succinamidyl (-NHC(O)CH 2 CH 2 In some embodiments, R is selected from the group consisting of ether, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), and sulfonate ester. N is H. In some embodiments, R N is C 1-6 In some embodiments, R N is C 1 In some embodiments, R N is C 2 In some embodiments, R N is C 3 In some embodiments, R N is C 4 In some embodiments, R N is C 5 In some embodiments, R N is C 6 In some embodiments, R N is carbonyl (-C(O)-). In some embodiments, R N is a carbamate (-NHC(O)O-). In some embodiments, R N is urea (-NHC(O)NH-). In some embodiments, R N is a disulfide (-SS-). N is an ether (-O-). In some embodiments, R N is succinyl (-(O)CCH 2 CH 2 In some embodiments, R N is succinamidyl (-NHC(O)CH 2 CH 2In some embodiments, R N is an ether. In some embodiments, R N is a carbonate (-OC(O)O-). In some embodiments, R N is succinoyl. In some embodiments, R N is a phosphate ester (-O-(O)POH-O-). In some embodiments, R N is a sulfonate ester.

[0043] In some embodiments, the lipid of the peptide-lipid conjugate is selected from didecyloxypropyl (C10), dilauryloxypropyl (C12), dimyristyloxypropyl (C14), dipalmityloxypropyl (C16), or distearyloxypropyl (C18), 1,2-dipalmityloxypropyl-3-amine (DOMG), 1,2-dimyristyloxypropylamine (DMG), 1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine (DLPE), dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE). In some embodiments, the lipid of the peptide-lipid conjugate is didecyloxypropyl (C10). In some embodiments, the lipid of the peptide-lipid conjugate is dilauryloxypropyl (C12). In some embodiments, the lipid of the peptide-lipid conjugate is dimyristyloxypropyl (C14). In some embodiments, the lipid of the peptide-lipid conjugate is dipalmityloxypropyl (C16). In some embodiments, the lipid of the peptide-lipid conjugate is distearyloxypropyl (C18). In some embodiments, the lipid of the peptide-lipid conjugate is 1,2-dimyristyloxypropyl-3-amine (DOMG). In some embodiments, the lipid of the peptide-lipid conjugate is 1,2-dimyristyloxypropylamine (DMG). In some embodiments, the lipid of the peptide-lipid conjugate is 1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine (DLPE). In some embodiments, the lipid of the peptide-lipid conjugate is dimyristoyl-phosphatidylethanolamine (DMPE). In some embodiments, the lipid of the peptide-lipid conjugate is dipalmitoyl-phosphatidylethanolamine (DPPE). In some embodiments, the lipid of the peptide-lipid conjugate is dipalmitoylphosphatidylcholine (DPPC).In some embodiments, the lipid of the peptide-lipid conjugate is dioleoyl-phosphatidylethanolamine (DOPE). In some embodiments, the lipid of the peptide-lipid conjugate is distearoyl-phosphatidylethanolamine (DSPE). In some embodiments, the lipid of the peptide-lipid conjugate is cholesterol or a cholesterol derivative.

[0044] In some embodiments, the peptide is conjugated at its C-terminus to a lipid of the peptide-lipid conjugate, and the amino group at the N-terminus of the peptide is conjugated to one or two C 1-6 In some embodiments, the N-terminus of the peptide is substituted with one or two alkyl or amide groups. In some embodiments, the N-terminus of the peptide is substituted with one or two C 1-6 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 1 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 2 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 3 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 4 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 5 In some embodiments, the N-terminus of the peptide is substituted with one or two C alkyl groups. 6 It is substituted with an alkyl group.

[0045] In some embodiments, the peptide is conjugated at its N-terminus to a lipid of the peptide-lipid conjugate, and the amino acid at the C-terminus of the peptide is alkylated to C 1-6 In some embodiments, the amino acid at the C-terminus of the peptide is amidated to form an alkyl ester. In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 1-6In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 1 In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 2 In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 3 In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 4 In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 5 In some embodiments, the amino acid at the C-terminus of the peptide is alkylated to form an alkyl ester. 6 Formation of alkyl esters.

[0046] In some embodiments, the lipid composition comprises 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium propane-(DODAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2 -Dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-Dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-Distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-Dimyrityloxyprop-3-yl)-N,N-dimethyl-N-hydroxy Ethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-oc-tadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl1-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOB) A), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and 2,The lipid composition further comprises one or more cationic lipids selected from 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (DLin-K-XTC2-DMA). In some embodiments, the lipid composition further comprises 5-carboxyspermylglycine dioctadecylamide (DOGS). In some embodiments, the lipid composition further comprises 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA). In some embodiments, the lipid composition further comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments, the lipid composition further comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the lipid composition further comprises 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA). In some embodiments, the lipid composition further comprises 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA). In some embodiments, the lipid composition further comprises 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). In some embodiments, the lipid composition further comprises N-dioleyl-N,N-dimethylammonium chloride (DODAC). In some embodiments, the lipid composition further comprises N,N-distearyl-N,N-dimethylammonium bromide (DDAB). In some embodiments, the lipid composition further comprises N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). In some embodiments, the lipid composition further comprises 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-oc-tadecadieneoxy)propane (CLinDMA). In some embodiments, the lipid composition further comprises N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA). In some embodiments, the lipid composition further comprises 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In some embodiments, the lipid composition further comprises 2,In some embodiments, the lipid composition further comprises 3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP). In some embodiments, the lipid composition further comprises 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP). In some embodiments, the lipid composition further comprises 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP). In some embodiments, the lipid composition further comprises 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA). In some embodiments, the lipid composition further comprises 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane. In some embodiments, the lipid composition further comprises 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (DLin-K-XTC2-DMA).

[0047] In some embodiments, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the one or more ionizable cationic lipids are selected from the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0048] In some embodiments, the lipid composition further comprises one or more helper lipids.

[0049] In some embodiments, the lipid composition further comprises a sterol. In some embodiments, the sterol is cholesterol.

[0050] In one embodiment, a lipid composition is provided comprising a peptide-lipid conjugate as provided herein, and also includes the embodiments and nucleic acids, i. the peptide of the peptide-lipid conjugate consists of about 4 to about 52 amino acids; ii. at least about 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline; iii. about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have hydrophilic side chains; iv. less than about 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine; v. the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol; vi. The peptide-lipid conjugate comprises from about 0.1 mol % to about 10 mol % of all lipids in the lipid composition.

[0051] In some embodiments, the nucleic acid is selected from siRNA, antisense oligonucleotide, UNA oligomer, mRNA, microRNA, and DNA. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the nucleic acid is a UNA oligomer. In some embodiments, the nucleic acid is microRNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is a self-replicating mRNA. In some embodiments, the nucleic acid is siRNA.

[0052] In some embodiments, a method of treating a disease in a subject in need thereof is provided, the method comprising administering a lipid composition of any one of the preceding claims. The disease may be cancer. The disease may be an autoimmune disease. The disease may be an inflammatory disease. The disease may be an infectious disease. In some embodiments, the disease is ornithine transcarbamylase deficiency (OTC deficiency). In some embodiments, the disease is cystic fibrosis (CF). In some embodiments, the disease is autoimmune hepatitis. In some embodiments, the disease is a liver viral disease (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E). In some embodiments, the disease is hepatitis B. In some embodiments, the disease is non-alcoholic steatohepatitis (NASH). In some embodiments, a method of expressing a protein or polypeptide of interest in a cell is provided, the method comprising contacting the cell with a lipid composition of the present disclosure.

[0053] In some embodiments, a vaccine is provided comprising the lipid composition of the present disclosure. In some embodiments, the vaccine is a SARS-CoV-2 vaccine. In some embodiments, the vaccine is an influenza vaccine. In some embodiments, the vaccine is an HIV vaccine. In some embodiments, the vaccine is an Ebola vaccine. In some embodiments, the vaccine is a cancer vaccine.

[0054] In one embodiment, a method of inducing an immune response in a subject is provided, comprising administering a vaccine to the subject.Thus, in some embodiments, a method of preventing disease in a subject is provided, comprising administering to the subject a lipid composition as provided herein, including embodiments thereof.

[0055] In some embodiments, a method of inhibiting expression of a gene or messenger RNA of interest in a cell is provided, comprising contacting the cell with a lipid composition of the present disclosure.

[0056] In some embodiments, a method is provided for expressing a protein or polypeptide of interest in a subject lacking the protein or polypeptide of interest, comprising administering to the subject a lipid composition of the present disclosure, wherein the lipid composition comprises mRNA encoding the protein or polypeptide of interest.

[0057] For the methods provided herein, in some embodiments, the lipid composition is administered intravenously or intramuscularly. In some embodiments, the lipid composition is administered intravenously. In some embodiments, the lipid composition is administered intramuscularly.

[0058] In some embodiments, the method for editing a gene in a cell comprises contacting the cell with the lipid composition of the present disclosure comprising mRNA, wherein the mRNA encodes a gene editing enzyme. In some embodiments, the gene editing enzyme comprises one or more components required for gene editing by zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or clustered regularly interspaced short palindromic repeats system (CRISPR / Cas). In some embodiments, the gene editing enzyme is zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, or clustered regularly interspaced short palindromic repeats system (CRISPR / Cas). In some embodiments, the gene editing enzyme is a TALEN enzyme.

[0059] In some embodiments, a method of delivering a nucleic acid to a cell is provided, the method comprising contacting the cell with a lipid composition provided herein, including embodiments thereof.

[0060] In some embodiments, a method of imaging a cell is provided, the method comprising contacting a cell with a lipid composition provided herein, including embodiments thereof, wherein the nucleic acid encodes a detectable protein.

[0061] In embodiments, methods of making the lipid compositions provided herein, including those embodiments, include: i) contacting a lipid nucleic acid with a peptide-lipid conjugate; and ii) allowing the peptide-lipid conjugate to encapsulate the nucleic acid.

[0062] Lipid-based formulations Therapies based on intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. In fact, naked nucleic acid materials cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, uptake by phagocytes, and their ability to activate immune responses, all features that hinder their clinical development. When exogenous nucleic acid materials (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and are removed from the blood circulation before having a chance to encounter target cells in or outside the vascular system. It has been reported that the half-life of naked nucleic acids in the bloodstream is approximately several minutes (Kawabata K, Takakura Y, Hashida MPharm Res.1995 Jun;12(6):825-30). Chemical modifications and proper delivery methods can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increases the stability and efficacy of nucleic acid-based therapies. In addition, RNA or DNA are anionic hydrophilic polymers that are also anionic on the surface, which is unfavorable for cellular uptake. Therefore, the success of nucleic acid-based therapy depends primarily on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0063] Furthermore, upon internalization into target cells, nucleic acid delivery vectors are challenged by intracellular barriers, including endosomal uptake, lysosomal degradation, unpackaging of the nucleic acid from the vector, translocation across the nuclear membrane (for DNA), and release in the cytoplasm (for RNA). Successful nucleic acid-based therapy therefore relies on the ability of the vector to deliver the nucleic acid to a target site inside the cell to obtain sufficient levels of the desired activity, such as gene expression.

[0064] Although some gene therapies can successfully utilize viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and simplicity of large-scale production. One of the most significant advances in lipid-based nucleic acid therapy occurred in August 2018, when patisiran (ALN-TTR02) was approved by the U.S. Food and Drug Administration (FDA) and the European Commission (EC) as the first siRNA therapeutic. ALN-TTR02 is an siRNA formulation based on the so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still under development.

[0065] Some art-recognized lipid formulation delivery vehicles for nucleic acid therapeutics include polymer-based carriers (such as polyethyleneimine (PEI), lipid nanoparticles and liposomes), nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions, according to various embodiments. Because these lipid formulations can vary in their structure and composition and can be expected in a rapidly developing field, the art uses several different terms to describe a single type of delivery vehicle. At the same time, the terminology of lipid formulations has changed with respect to their intended meaning throughout the scientific literature, and this inconsistent use has caused confusion regarding the exact meaning of some terms of lipid formulations. Among several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail for the purposes of this disclosure and are defined herein.

[0066] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is typically formed by amphiphilic molecules such as lipids of synthetic or natural origin that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally exist as spherical vesicles and can range in size from 20 nm to several microns. Liposome formulations can be prepared as colloidal dispersions or lyophilized to reduce stability risks and improve the shelf life of liposomal drugs. Methods for preparing liposome compositions are known in the art and are within the skill of the artisan.

[0067] Liposomes with only one bilayer are called unilamellar, and liposomes with two or more bilayers are called multilamellar. The most common types of liposomes are small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs). In contrast to liposomes, lysosomes, micelles, and reverse micelles are composed of a single layer of lipid. Generally, liposomes are considered to have a single internal compartment, but some formulations can be multivesicular liposomes (MVLs), which consist of multiple discontinuous internal aqueous compartments separated by several non-concentric lipid bilayers.

[0068] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, considering that liposomes are essentially analogues of biological membranes and can be prepared from both natural and synthetic phospholipids (Int. J. Nanomedicine. 2014;9:1833-1843). In their use as drug delivery vehicles, liposomes have an aqueous core surrounded by a hydrophobic membrane, so that hydrophilic solutes dissolved in the core cannot easily pass through the bilayer, and hydrophobic compounds associate with the bilayer. Thus, liposomes can be loaded with hydrophobic and / or hydrophilic molecules. When liposomes are used to carry nucleic acids such as RNA, the nucleic acid is contained within the liposomal compartment in the aqueous phase.

[0069] Cationic Liposomes Liposomes can be composed of cationic lipids, anionic lipids, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes made entirely or partially from positively charged lipids, more specifically, lipids that contain both cationic groups and lipophilic moieties. In addition to the general characteristics described above for liposomes, the positively charged portion of the cationic lipids used in cationic liposomes offers some advantages and some unique structural features. For example, the lipophilic portion of cationic lipids is hydrophobic and thus orients itself away from the aqueous interior of the liposome and associates with other non-polar and hydrophobic species. Conversely, the cationic portion associates with polar molecules and species that can complex with aqueous media and, more importantly, with the aqueous interior of cationic liposomes. For these reasons, cationic liposomes are increasingly being investigated for use in gene therapy due to their preference for negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical use. Cationic lipids suitable for use in cationic liposomes are listed below.

[0070] Lipid Nanoparticles In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in the solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase in their interior, but rather, lipids from the bilayer or monolayer shell are directly complexed to the internal compound, thereby encapsulating it within the solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform distribution of shapes and sizes. Sources differ as to the size that qualifies a lipid particle as a nanoparticle, but there is some overlapping agreement that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. However, more commonly, they are considered to be smaller than 120 nm, or even smaller than 100 nm.

[0071] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include ionic cationic lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. Ionic cationic lipids with an apparent pKa value of less than about 7 have the advantage that they can complex with the negatively charged backbone of the nucleic acid, providing the cationic lipid for loading into the lipid nanoparticle at pH values ​​that are less than the pKa of the positively charged ionized lipid. Then, at physiological pH values, the lipid nanoparticles can conform a relatively neutral exterior, allowing for a significant increase in the circulatory half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, strong transfection, improved tissue penetration for delivering therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0072] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids have been widely investigated as synthetic materials for the delivery of nucleic acid drugs. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and were also characterized by a wide size distribution ranging from the submicron scale to several microns. Lipoplexes such as Lipofectamine® reagent have found considerable utility for in vitro transfection. However, these first generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by cationic lipids) result in rapid plasma clearance, hemolysis, and other toxicities, as well as immune system activation.

[0073] Lipid-nucleic acid formulations The nucleic acid, or a pharma- ceutically acceptable salt thereof, can be incorporated into a lipid formulation (ie, a lipid-based delivery vehicle).

[0074] In the context of the present disclosure, the lipid-based delivery vehicle typically functions to transport a desired nucleic acid (such as siRNA, plasmid DNA, mRNA, self-replicating RNA, etc.) to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing a nucleic acid. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or bilayer of lipid molecules and nucleic acid. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or polymer. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, the lipid formulation preferably encapsulates a nucleic acid.

[0075] The present disclosure provides lipid formulations comprising one or more therapeutic nucleic acid molecules encapsulated in lipid formulations.In some embodiments, the lipid formulation comprises liposomes.In some embodiments, the lipid formulation comprises cationic liposomes.In some embodiments, the lipid formulation comprises lipid nanoparticles.

[0076] In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid formulation, which makes the nucleic acid in the lipid formulation resistant to nuclease degradation in aqueous solution. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals, such as humans.

[0077] The lipid formulations of the present disclosure also typically have a total lipid:nucleic acid ratio (mass / mass ratio) of about 1:1 to about 100:1, about 1:1 to about 50:1, about 2:1 to about 45:1, about 3:1 to about 40:1, about 5:1 to about 38:1, about 6:1 to about 40:1, about 7:1 to about 35:1, about 8:1 to about 30:1, about 10:1 to about 25:1, about 8:1 to about 12:1, about 13:1 to about 17:1, about 18:1 to about 24:1, or about 20:1 to about 30:1. In some preferred embodiments, the total lipid:nucleic acid ratio (mass / mass ratio) is about 10:1 to about 25:1. The ratio may be any value or subvalue within the recited range, including the endpoints.

[0078] The lipid formulations of the present disclosure typically have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 70 nm, ...80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, The lipid nanoparticles have an average diameter of about 0 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited range, including the end points. In addition, the nucleic acid is resistant in aqueous solution to degradation by nucleases when present in the lipid nanoparticles of the present disclosure.

[0079] In a preferred embodiment, the lipid formulation comprises a nucleic acid, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits particle aggregation (e.g., one or more PEG-lipid conjugates and / or peptide-lipid conjugates of the present disclosure).The lipid formulation can also comprise cholesterol.

[0080] In nucleic acid lipid formulations, nucleic acid may be completely encapsulated in the lipid portion of the formulation, thereby protecting nucleic acid from nuclease degradation.In a preferred embodiment, the lipid formulation containing nucleic acid is completely encapsulated in the lipid portion of the lipid formulation, thereby protecting nucleic acid from nuclease degradation.In certain cases, the nucleic acid in the lipid formulation is not substantially degraded after the particle is exposed to nuclease at 37°C for at least 20, 30, 45, or 60 minutes.In certain other cases, the nucleic acid in the lipid formulation is not substantially degraded after the formulation is incubated in serum at 37°C for at least 30, 45, or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.In other embodiments, nucleic acid is complexed with the lipid portion of the formulation.

[0081] In the context of nucleic acids, complete encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye whose fluorescence is enhanced when associated with nucleic acid. Encapsulation is determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of detergent.

[0082] In other embodiments, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-lipid nanoparticles.

[0083] In some embodiments, the lipid formulation comprises nucleic acid that is fully encapsulated within the lipid portion of the formulation, such that the nucleic acid occupies about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 3 ...50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 95%, about 50% to about 9 0%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction or range therein) have nucleic acid encapsulated therein. The amount may be any value or subvalue within the recited range, including the endpoints.

[0084] Depending on the intended use of the lipid formulation, the ratio of the components can be varied and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0085] According to some embodiments, the expressible polynucleotide, the nucleic acid active agent, and the mRNA construct can be lipid-formulated. The lipid formulation is preferably selected from, but not limited to, a liposome, a cationic liposome, and a lipid nanoparticle. In a preferred embodiment, the lipid formulation is a cationic liposome or a lipid nanoparticle (LNP); (a) a nucleic acid (e.g., mRNA, siRNA, etc.); (b) a cationic lipid; (c) a peptide-lipid conjugate of the present disclosure; and (d) optionally a non-cationic lipid (such as a neutral lipid); and (e) optionally, a sterol.

[0086] In some embodiments, the cationic lipid is an ionic cationic lipid. In one embodiment, the lipid nanoparticle formulation comprises (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a peptide-lipid conjugate of the present disclosure, in a molar ratio of about 20% to about 40% ionic cationic lipid: about 25% to about 45% helper lipid: about 25% to about 45% sterol; about 0.5 to 5% peptide-lipid conjugate. Examples of cationic lipids (including ionic cationic lipids), helper lipids (e.g., neutral lipids), and sterols are described below.

[0087] Cationic lipids The lipid formulation preferably comprises cationic lipid suitable for forming cationic liposome or lipid nanoparticle. Cationic lipids have been widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. In general, cationic lipids are amphiphilic substances that contain a positive hydrophilic head group, two (or more) lipophilic tails or steroid moieties, and a connector between these two domains. Preferably, cationic lipids carry a net positive charge at approximately physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-methylammonium sulfate) can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, providing high in vitro transfection efficiency.

[0088] In the lipid formulations of the present disclosure, the cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salts), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salts), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleo ... propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-di Linoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l, 2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydro (2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28 31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-l-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-l-amine (MC4 ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), l,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,Cationic lipids include, but are not limited to, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). In addition, commercially available preparations of cationic lipids can be used, such as Lipofectin (including DOTMA and DOPE available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE available from GIBCO / BRL).

[0089] Other suitable cationic lipids are described in International Publication Nos. WO09 / 086558, WO09 / 127060, WO10 / 048536, WO10 / 054406, WO10 / 088537, WO10 / 129709, and WO2011 / 153493, U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803, U.S. Patent No. 8,158,601, and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0090] Other suitable cationic lipids include those with alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids called amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids with less saturated alkyl chains are easier to size for filter sterilization purposes, especially when the complexes must be sized to less than about 0.3 microns. Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of C14-C22 may be used. Other scaffolds may also be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0091] In some embodiments, the lipid formulation comprises a cationic lipid having formula I as described in patent application PCT / EP2017 / 064066, the disclosure of which is also incorporated herein by reference in this context.

[0092] In some embodiments, the amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species, and not all of the lipids need be present in a charged or neutral form. Lipids that have two or more protonatable or deprotonatable groups or are zwitterionic are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group ranging from about 4 to about 11. In some embodiments, the ionic cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionic cationic lipid is about 6 to about 7.

[0093] In some embodiments, the lipid formulation comprises an ionic cationic lipid of formula (I): [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 5 and R 6 is a straight or branched chain C 1 -C 31 Alkyl, C 2 -C 31 Alkenyl or C 2 -C 31 L is independently selected from the group consisting of alkynyl and cholesteryl; 5 and L 6 is a linear C 1 -C 20 Alkyl and C 2 -C20 alkenyl; 5 is -C(O)O-, which gives -C(O)OR 6 is formed or -OC(O)-, whereby -OC(O)-R 6 is formed, and X 6 is -C(O)O-, which gives -C(O)OR 5 is formed or -OC(O)-, whereby -OC(O)-R 5 is formed, and X 7 is S or O, and L 7 is absent or is lower alkyl, R 4 is a linear or branched chain C 1 -C 6 alkyl, and R 7 and R 8 is hydrogen and straight or branched chain C 1 -C 6 alkyl.

[0094] In some embodiments, X 7 is S.

[0095] In some embodiments, X 5 is -C(O)O-, thereby -C(O)OR 6 is formed, and X 6 is -C(O)O-, thereby -C(O)OR 5 is formed.

[0096] In some embodiments, R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0097] In some embodiments, L 5 and L 6 are each independently 1 -C 10 In some embodiments, L is alkyl. 5 is C 1-C 3 alkyl and L6 is C 1 -C 5 In some embodiments, L is alkyl. 6 is C 1 -C 2 In some embodiments, L is alkyl. 5 and L 6 are linear C 7 In some embodiments, L is alkyl. 5 and L 6 are linear C 9 It is an alkyl.

[0098] In some embodiments, R 5 and R 6 is, independently at each occurrence, alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C 2 -C 9 In some embodiments, the alkenyl contains a single double bond. In some embodiments, R 5 and R 6 are each alkyl. In some embodiments, R 5 is a branched alkyl. In some embodiments, R 5 and R 6 is C 9 Alkyl, C 9 Alkenyl, and C 9 alkynyl. In some embodiments, R 5 and R 6 are each independently 11 Alkyl, C 11 Alkenyl, and C 11 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 is C 7 Alkyl, C 7 Alkenyl, and C 7 alkynyl. In some embodiments, R 5 is -CH((CH 2 )p CH 3 ) 2 or -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3 ), where p is 4 to 8. In some embodiments, p is 5 and L 5 is C 1 -C 3 In some embodiments, p is 6 and L is alkyl. 5 is C 3 In some embodiments, p is 7. In some embodiments, p is 8 and L is alkyl. 5 is C 1 -C 3 In some embodiments, R 5 is -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3 ) wherein p is 7 or 8.

[0099] In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene.

[0100] In some embodiments, L 7 does not exist, R 4 is ethylene, and X 7 is S and R 7 and R 8 and each is methyl. 7 does not exist, R 4 is n-propylene, and X 7 is S and R 7 and R 8 and each is methyl. 7 does not exist, R 4 is ethylene, and X 7is S and R 7 and R 8 are each ethyl.

[0101] In some embodiments, X 7 is S and X 5 is -C(O)O-, and therefore -C(O)OR 6 is formed, and X 6 is -C(O)O-, and therefore -C(O)OR 5 is formed, and L 5 and L 6 are each independently linear C 3 -C 7 is alkyl, and L 7 does not exist, and R 5 -CH(CH 2 ) p CH 3 ) 2 and R 6 is C 7 -C 12 In some further embodiments, p is 6 and R 6 is C 9 It is alkenyl.

[0102] In some embodiments, the lipid formulation comprises an ionic cationic lipid selected from the group of ATX lipids disclosed hereinabove.

[0103] In some embodiments, any one or more of the lipids listed herein may be explicitly excluded.

[0104] Helper lipids and sterols The mRNA lipid formulation of the present disclosure can include a helper lipid, which can be referred to as a neutral lipid, a neutral helper lipid, a non-cationic lipid, a non-cationic helper lipid, an anionic lipid, an anionic helper lipid, or a zwitterionic lipid. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to have increased cellular uptake when helper lipids are present in the formulation. (Curr.Drug Metab.2014;15(9):882-92). For example, some studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), di-oleoyl-phosphatidyl-ethanoalamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), are more fusogenic (i.e., facilitate fusion) than cationic lipids, and may affect the polymorphic characteristics of lipid-nucleic acid complexes, promoting the transition from lamellar to hexagonal phases, thus inducing fusion and disruption of cell membranes. (Nanomedicine (Lond). 2014 Jan; 9 (1): 105-20). In addition, the use of helper lipids may help reduce any potential adverse effects of using many common cationic lipids, such as toxicity and immunogenicity.

[0105] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine ...DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine ( Examples of phosphatidylethanolamine include oleyl-phosphatidylethanolamine (POPE), palmitoyloleyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably C 10 -C 24 It is an acyl group derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0106] Additional examples of non-cationic lipids include sterols such as cholesterol and its derivatives. One study concluded that as a helper lipid, cholesterol increases the charge spacing of the lipid layer that matches the nucleic acid, making the charge distribution more closely match that of the nucleic acid. (JRSoc.Interface.2012 Mar 7;9(68):548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0107] In some embodiments, the helper lipid present in lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In other embodiments, the helper lipid present in lipid formulation comprises or consists of one or more phospholipids, for example, cholesterol-free lipid formulations.In still other embodiments, the helper lipid present in lipid formulation comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid formulations.

[0108] Other examples of helper lipids include non-phosphorus containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0109] In some embodiments, the helper lipid comprises about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range thereof) of the total lipid present in the lipid formulation.

[0110] In some embodiments, the total amount of helper lipids in the formulation includes two or more helper lipids, and the total amount of helper lipids includes about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range thereof) of the total lipids present in the lipid formulation. In some embodiments, the helper lipid is a combination of DSPC and DOTAP. In some embodiments, the helper lipid is a combination of DSPC and DOTMA.

[0111] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.

[0112] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ±5 mol %.

[0113] A lipid formulation containing a cationic lipid compound or an ionic cationic lipid compound may be about 20-40% cationic lipid compound, about 25-40% cholesterol, about 25-50% helper lipid, and about 0.5-5% peptide-lipid conjugate of the present disclosure, on a molar basis, the percentages being of the total lipid present in the formulation. In some embodiments, the composition is about 22-30% cationic lipid compound, about 30-40% cholesterol, about 30-40% helper lipid, and about 0.5-3% peptide-lipid conjugate of the present disclosure, the percentages being of the total lipid present in the formulation.

[0114] Lipid conjugates In some embodiments, one or more peptide-lipid conjugates of the present disclosure comprise about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fraction or range thereof) of the total lipid present in the lipid formulation. In other embodiments, the one or more peptide-lipid conjugates comprise about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5% (or any fraction or range thereof) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited range, including the endpoints.

[0115] The percentage of peptide-lipid conjugate present in the lipid formulation of the present disclosure is a target amount, and the actual amount of peptide-lipid conjugate present in the formulation may vary, for example, by ±0.5 mol%. Those skilled in the art will understand that the concentration of lipid conjugate may vary depending on the lipid conjugate used and the rate at which the lipid formulation becomes fusogenic.

[0116] Mechanisms of action for cellular uptake of lipid formulations Lipid formulations for intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating the target cell by utilizing the endocytosis mechanism of the target cell, where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157, 2018). Specifically, in the case of the nucleic acid-lipid formulations described herein, the lipid formulation enters the cell via receptor-mediated endocytosis. Prior to endocytosis, functionalized ligands, such as the peptide-lipid conjugates of the present disclosure, on the surface of the lipid delivery vehicle can be shed from the surface, which triggers internalization into the target cell. During endocytosis, a portion of the cell's plasma membrane surrounds the vector and engulfs it into a vesicle, which then pinches the vesicle out of the cell membrane, enters the cytosol, and finally goes through the endolysosomal pathway. For delivery vehicles containing ionic cationic lipids, the increased acidity as endosomes age results in vehicles with strong positive charges on the surface. The interaction between the delivery vehicle and the endosomal membrane then results in a membrane fusion event that leads to cytoplasmic delivery of the payload. For mRNA or self-replicating RNA payloads, the cell's own internal translation process then translates the RNA into the encoded protein. The encoded protein can undergo further post-translational processing, including transport to the target organelle or intracellular location.

[0117] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate is exchanged from lipid formulation and then the rate at which lipid formulation becomes fusogenic can be controlled.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which lipid formulation becomes fusogenic.Other methods that can be used to control the rate at which lipid formulation becomes fusogenic will be clear to those skilled in the art upon reading this disclosure.Also, by controlling the composition and concentration of lipid conjugate, the size of liposome or lipid particle can be controlled.

[0118] Lipid formulation manufacturing There are many different methods for the preparation of lipid formulations containing nucleic acids. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, double asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.

[0119] Thin Film Hydration In the thin film hydration method (TFH) or Bangham method, lipids are dissolved in an organic solvent and then evaporated through the use of a rotary evaporator, resulting in the formation of a thin lipid layer. After layer hydration with an aqueous buffer solution containing the compound to be loaded, multilamellar vesicles (MLVs) are formed, which can be reduced in size to generate small unilamellar vesicles (LUVs) or large unilamellar vesicles (SUVs) by extrusion through a membrane or by sonication of the starting MLVs.

[0120] Double Emulsion Lipid formulations can also be prepared through a double emulsion technique, which involves dissolving lipids in a water / organic solvent mixture. An organic solution containing water droplets is mixed with an excess of aqueous medium, resulting in the formation of a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, some of the water droplets collapse, resulting in large unilamellar vesicles (LUVs).

[0121] Reverse Phase Evaporation The reverse phase evaporation (REV) method can also achieve nucleic acid loaded LUVs. In this technique, a two-phase system is formed by dissolving phospholipids in an organic solvent and an aqueous buffer. The resulting suspension is then sonicated for a short period of time until the mixture becomes a clear one-phase dispersion. The lipid formulation is achieved after evaporation of the organic solvent under reduced pressure. This technique is used to encapsulate different large and small hydrophilic molecules, including nucleic acids.

[0122] Microfluidic preparation Microfluidic methods, unlike other bulk techniques, give the possibility to control the lipid hydration process. Methods can be classified into continuous-flow microfluidics and droplet-based microfluidics according to the way the flows are manipulated. In the microhydrodynamic focusing (MHF) method, operating in continuous-flow mode, lipids are dissolved in isopropyl alcohol, which is hydrodynamically focused in a microchannel cross-junction between two aqueous buffer streams. The vesicle size can be controlled by regulating the flow rate, and thus the lipid solution / buffer dilution process. The method can be used to generate oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlet ports and one outlet port.

[0123] Double asymmetric centrifugation Double asymmetric centrifugation (DAC) differs from more common centrifugation because it uses an additional rotation around its own vertical axis. Efficient homogenization is achieved by the two overlay movements created; that is, the sample is pushed outward as in a normal centrifuge, and then pushed toward the center of the vial by an additional rotation. By mixing the lipid and NaCl solutions, a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be adjusted by optimizing the DAC speed, lipid concentration, and homogenization time.

[0124] Ethanol injection The ethanol injection (EI) method can be used for nucleic acid encapsulation. This method provides for the rapid injection of an ethanol solution in which lipids are dissolved into an aqueous medium containing the nucleic acid to be encapsulated through the use of a needle. When the phospholipids are dispersed throughout the medium, vesicles spontaneously form.

[0125] Detergent dialysis Detergent dialysis method can be used to encapsulate nucleic acid.In brief, lipid and plasmid are solubilized in detergent solution of appropriate ionic strength, and after removing detergent by dialysis, stabilized lipid formulation is formed.Unencapsulated nucleic acid is then removed by ion exchange chromatography, and vesicles are emptied by sucrose density gradient centrifugation.This technique is very sensitive to cationic lipid content and salt concentration of dialysis buffer, and this method is also difficult to scale up.

[0126] Spontaneous vesicle formation upon ethanol dilution Stable lipid formulations can also be generated via the spontaneous vesicle formation by ethanol dilution method, in which stepwise or dropwise ethanol dilution provides for the spontaneous formation of nucleic acid-loaded vesicles by the controlled addition of lipids dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.

[0127] Pharmaceutical Compositions and Delivery Methods In order to promote in vivo nucleic acid activity (e.g., mRNA expression, or knockdown by ASO or siRNA), the delivery vehicle of the nucleic acid lipid formulation described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or with a pharmacological composition that is mixed with suitable excipients.The techniques of drug formulation and administration can be found in "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa, latest edition).

[0128] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed according to current medical practice, taking into consideration the subject's clinical condition, the site and method of administration, the administration schedule, the subject's age, sex, weight, and other factors relevant to the skilled artisan. An "effective amount" for purposes herein may be determined by experimental clinical studies, pharmacological, clinical, and relevant considerations as known to those skilled in the art of medical technology. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement, or elimination of symptoms and other indicators selected by the skilled artisan as an appropriate measure of disease progression, regression, or improvement. For example, an appropriate amount and administration regimen is one that causes at least transient production of a protein (e.g., an enzyme). Another example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom(s) of a disease, which may also be referred to as a "therapeutically effective amount." A "reduction" of a symptom(s) (and grammatical equivalents of this phrase) refers to a decrease in the severity or frequency of the symptom(s), or the elimination of the symptom(s). A "prophylactically effective amount" of an agent is an amount of the agent that, when administered to a subject, has the intended prophylactic effect, e.g., prevents or delays the onset (or recurrence) of an injury, disease, pathology, or condition, or reduces the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or symptoms thereof. A complete prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0129] The pharmaceutical compositions described herein can be inhalable compositions.Suitable routes of administration include, for example, intratracheal, inhalation, or intranasal.In some embodiments, administration results in delivery of nucleic acid to lung epithelial cells.In some embodiments, administration shows selectivity for lung epithelial cells over other types of lung cells and airway cells.

[0130] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the nucleic acid), (4) modify biodistribution (e.g., targeting the nucleic acid to a particular tissue or cell type), (5) increase the activity of the nucleic acid or protein expressed therefrom in vivo, and / or (6) alter the release profile of the nucleic acid or encoded protein in vivo.

[0131] Preferably, lipid formulations can be administered in a localized manner rather than systemically.Local delivery can affect in various ways depending on the tissue targeted.For example, the aerosol containing the composition of the present disclosure can be inhaled (for nose, trachea, or bronchial delivery).

[0132] Pharmaceutical compositions can be administered to any desired tissue.In some embodiments, the nucleic acid delivered by lipid formulations or compositions of the present disclosure is active in the tissue to which lipid formulations and / or compositions are administered.In some embodiments, the nucleic acid is active in tissues different from the tissue to which lipid formulations and / or compositions are administered.Examples of tissues that nucleic acid can be delivered include, but are not limited to, lung, trachea, and / or nasal cavity, muscle, liver, eye, or central nervous system.

[0133] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association an active ingredient (i.e., a nucleic acid) with an excipient and / or one or more other accessory ingredients. Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0134] Pharmaceutical compositions may additionally contain pharma- ceutically acceptable excipients, which as used herein include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, appropriate for the particular dosage form desired.

[0135] In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, but are not limited to, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA constructs, or mRNA (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0136] Thus, the formulations described herein can include one or more excipients in an amount that together increase the stability of the nucleic acid in the lipid formulation, increase cell transfection with the nucleic acid (e.g., mRNA or siRNA), increase expression of the encoded protein, and / or alter the release profile of the encoded protein, or increase knockdown of the target native nucleic acid. Additionally, the nucleic acid may be formulated using self-assembling nucleic acid nanoparticles.

[0137] Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated herein by reference in its entirety).The use of conventional excipient vehicles may be contemplated within the scope of the embodiments of the present disclosure, except insofar as any conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0138] The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in liquid before administration.The solid can be administered as a powder.In some embodiments, the pharmaceutical composition comprises a lyophilized nucleic acid-lipid formulation.

[0139] In a preferred embodiment, the dosage form of the pharmaceutical composition described herein can be a liquid suspension of the nucleic acid-lipid nanoparticles described herein. In some embodiments, the liquid suspension is in a buffer solution. In some embodiments, the buffer solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffer solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a combination of a sugar and glycerol, or a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature less than about -70°C. In some embodiments, the suspension is diluted with sterile water prior to inhalable administration. In some embodiments, inhalable administration comprises diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, the lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in a buffer solution described herein.

[0140] The compositions and methods of the present disclosure may be administered to a subject by various mucosal administration modes, including intranasal and / or intrapulmonary. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, and / or oral. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically actuated, or other types of actuators.

[0141] The compositions of the present disclosure may be administered in aqueous solution as a nasal or pulmonary spray, or may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved by administering the compositions in the form of droplets, particles, or sprays, which can be, for example, aerosolized, atomized, or nebulized. The particles of the composition, spray, or aerosol can be either liquid or solid form, for example, lyophilized lipid formulations. A preferred system for dispensing liquids as nasal sprays is disclosed in U.S. Patent No. 4,511,069. Such formulations can be conveniently prepared by dissolving the compositions according to the present disclosure in water to produce an aqueous solution, and sterilizing the solution. The formulations may be presented, for example, in a multi-drug container in a sealed dispensing system disclosed in U.S. Patent No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, YW Hien ed., Elsevier Publishers, New York, 1985 and U.S. Patent No. 4,778,810. Additional aerosol delivery forms may include, for example, pneumatic, jet, ultrasonic, and piezoelectric nebulizers, which deliver the nucleic acid lipid formulation or are suspended in a pharmaceutical solvent, for example, water, ethanol, or mixtures thereof.

[0142] The nasal and pulmonary spray solutions of the present disclosure typically include a surface active agent, such as a non-ionic surfactant (e.g., polysorbate-80), and the nucleic acid optionally formulated with one or more buffers, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In some embodiments of the present disclosure, the nasal spray solution further includes a propellant. The pH of the nasal spray solution may be pH 6.8 to 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer with a pH of 4 to 6. Other ingredients, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0143] In some embodiments, the present disclosure provides a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0144] The dosage form of the composition of the present disclosure can be in the form of a liquid, droplets or emulsion, or in the form of an aerosol.

[0145] The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in a liquid before administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0146] To formulate a composition for pulmonary delivery within the present disclosure, the nucleic acid-lipid formulation can be combined with various pharma- ceutically acceptable additives, as well as bases or carriers for dispersing the nucleic acid-lipid formulation(s). Examples of additives include pH adjusters, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is liquid, the tonicity of the formulation, measured with reference to the tonicity of a 0.9% (w / v) saline solution taken as 1, is typically adjusted to a value that does not induce substantially irreversible tissue damage to the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0147] The nucleic acid-lipid formulation may be dispersed in a base or vehicle, which may include a hydrophilic compound capable of dispersing the nucleic acid-lipid formulation and any desired additives.The base may be selected from a wide range of suitable carriers, including, but not limited to, copolymers of polycarboxylic acids or their salts, carboxylic acid anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and their non-toxic metal salts.Biodegradable polymers are often selected as bases or carriers, such as, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and improved structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking, etc. Carriers are provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers in this context may result in enhanced absorption of the nucleic acid-lipid formulation.

[0148] Alternatively, the compositions of the present disclosure may contain pharma- ceutically acceptable carrier materials required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional non-toxic pharma-ceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0149] In certain embodiments of the present disclosure, nucleic acid-lipid formulations may be administered in time-release formulations, for example, compositions that contain slow-release polymers.Nucleic acid-lipid formulations can be prepared with carriers that protect against rapid release, such as controlled release vehicles, for example, polymers, microencapsulated delivery systems, or bioadhesive gels.Long-term delivery of nucleic acid-lipid formulations in various compositions of the present disclosure can be achieved by including them in compositions that delay absorption, for example, aluminum monostearate hydrogels and gelatin.

[0150] Nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the nucleic acid composition, inhalation of an aerosol mist generated by a liquid nebulizer, or by use of dry powder equipment such as that described in U.S. Pat. No. 5,780,014, which is incorporated herein by reference.

[0151] In certain embodiments, the compositions of the present disclosure may be formulated such that they may be aerosolized or otherwise delivered as particulate liquids or solids prior to or upon administration to a subject. Such compositions may be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions, etc.) to generate particles that are easily respirable or inhalable by a subject. In some embodiments, such devices (e.g., metered dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers, or pneumoperitoneum devices) facilitate administration of a predetermined mass, volume, or dose (e.g., about 0.010 to about 0.5 mg / kg of nucleic acid per dose) of the composition to a subject. For example, in certain embodiments, the compositions of the present disclosure are administered to a subject using a metered dose inhaler that contains a suspension or solution comprising the composition and a suitable propellant. In certain embodiments, the compositions of the present disclosure may be formulated as particulate powders (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the present disclosure formulated as respirable particles are appropriately sized (e.g., average D50 or D90 particle size of about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or less) so that they may be respirable by a subject or delivered using a suitable device. In yet other embodiments, compositions of the present disclosure are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).In some embodiments, compositions of the present disclosure provide a dose of at least 0.010 mg / kg, at least 0.015 mg / kg, at least 0.020 mg / kg, at least 0.025 mg / kg, at least 0.030 mg / kg, at least 0.035 mg / kg, at least 0.040 mg / kg, at least 0.045 mg / kg, at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least In some embodiments, the subject is administered a single dose such that a concentration of at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg is administered in a single dose. In some embodiments, compositions of the present disclosure are administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of nucleic acid is administered in one or more doses.

[0152] In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject once a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject twice a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject three times a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject four times a month.

[0153] According to the present disclosure, a therapeutically effective dose of the provided composition, when administered periodically, results in an increase in nucleic acid activity levels in a subject, compared to baseline activity levels before treatment. Typically, activity levels are measured in biological samples obtained from a subject, such as blood, plasma or serum, urine, or solid tissue extracts. Baseline levels can be measured immediately before treatment. In some embodiments, administering the pharmaceutical compositions described herein results in an increase in nucleic acid activity levels in biological samples (e.g., plasma / serum or lung epithelial swabs) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels before treatment. In some embodiments, administration of provided compositions results in an increase in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to pre-treatment baseline levels for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.

[0154] definition At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include any and all individual subcombinations of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" includes methyl, ethyl, C 3 Alkyl, C4 Alkyl, C 5 Alkyl, and C 6 It is specifically intended to disclose alkyl individually.

[0155] The phrases "administered in combination with" or "administration in combination with" mean that two or more agents are administered to a subject simultaneously or within an interval such that there may be an overlap in the effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minutes of each other. In some embodiments, administration of the agents is spaced sufficiently close to each other that a combined effect (e.g., a synergistic effect) is achieved.

[0156] As used herein, the term "administration" refers to oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In some embodiments, administration does not include administration of any active agent other than the listed active agents.

[0157] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0158] The term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of the possible values).

[0159] The terms "associated," "conjugated," "bonded," "attached," and "tethered," when used in reference to two or more moieties, mean that the moieties are physically associated or bound to one another, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under conditions in which the structure is used, such as physiological conditions. An "association" need not necessarily be strictly a direct chemical covalent bond. Ionic or hydrogen bonds, or hybridization-based connections may also suggest that the entities that are "associated" are sufficiently stable to remain physically associated.

[0160] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or clear from the context. A claim or description containing "or" between one or more group members is deemed satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0161] As used herein, the term "acyl" refers to a hydrogen or alkyl group (e.g., a haloalkyl group), as defined herein, which is attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl, and the like. Examples of unsubstituted acyl groups include 1 to 7, 1 to 11, or 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents, as described herein.

[0162] As used herein, the term "alkenyl," unless otherwise specified, refers to a monovalent straight or branched chain group of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds, and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyl includes both cis and trans isomers. Alkenyl groups may be optionally substituted with 1, 2, 3, or 4 substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0163] The term "alkoxy" refers to a chemical substituent of formula -OR, where R is, unless otherwise specified, C 1 -20 alkyl group (e.g., C 1-6 or C 1-10 Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents (e.g., hydroxy or alkoxy) as defined herein.

[0164] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group. Examples of unsubstituted alkoxyalkyl groups include alkyl groups having 2 to 40 carbons (e.g., 2 to 12 or 2 to 20 carbons, e.g., C 1-6 Alkoxy-C 1-6 Alkyl, C 1-10 Alkoxy-C 1-10 Alkyl, or C 1-20 Alkoxy-C 1-20 In some embodiments, alkyl and alkoxy can each be further substituted with 1, 2, 3, or 4 substituents as defined herein for each respective group.

[0165] As used herein, the term "alkoxycarbonyl" refers to alkoxy, as defined herein, and includes a carbonyl atom (e.g., -C(O)-OR, where R is H, or an optionally substituted C 1-6 , C 1-10 , or C 1-20 The alkoxycarbonyl is attached to the parent molecular group via an alkyl group. Examples of unsubstituted alkoxycarbonyls include 1 to 21 carbons (e.g., 1 to 11 or 1 to 7 carbons). In some embodiments, the alkoxy group is further substituted with 1, 2, 3, or 4 substituents described herein.

[0166] As used herein, the term "alkoxycarbonylalkyl" refers to an alkyl group, as defined herein, substituted with an alkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)-OR, where R is an optionally substituted C 1-20 , C 1-10 , or C 1-6 Examples of unsubstituted alkoxycarbonylalkyl groups include those having 3 to 41 carbons (e.g., 3 to 10, 3 to 13, 3 to 17, 3 to 21, or 3 to 31 carbons, e.g., C 1-6 Alkoxycarbonyl-C 1-6 Alkyl, C 1-10 Alkoxycarbonyl-C 1-10 Alkyl, or C 1-20 Alkoxycarbonyl-C 1-20 In some embodiments, each alkyl and alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents described herein (e.g., hydroxy groups).

[0167] As used herein, the term "alkoxycarbonylalkenyl" refers to an alkenyl group, as defined herein, including an alkoxycarbonyl group, as defined herein (e.g., -alkenyl-C(O)-OR, where R is an optionally substituted C 1-20 , C 1-10 , or C 1-6Examples of unsubstituted alkoxycarbonylalkenyls include those having 4 to 41 carbons (e.g., 4 to 10, 4 to 13, 4 to 17, 4 to 21, or 4 to 31 carbons, e.g., C 1-6 Alkoxycarbonyl-C 2-6 Alkenyl, C 1-10 Alkoxycarbonyl-C 2-10 Alkenyl, or C 1-20 Alkoxycarbonyl-C 2-20 In some embodiments, each alkyl, alkenyl, and alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents described herein (e.g., hydroxy groups).

[0168] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range, e.g., "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also encompasses occurrences of the term "alkyl" where no numerical range is specified). The alkyl group may also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may also be a "C 1-4 By way of example only, "C 1-4 "Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited in any manner to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0169] The term "lower alkyl" means a group having 1 to 6 carbons in the chain which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0170] As used herein, the term "alkylsulfinyl" refers to an alkyl group attached to the parent molecular group via an -S(O)- group. Examples of unsubstituted alkylsulfinyl groups are 1 to 6, 1 to 10, or 1 to 20 carbons. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein.

[0171] As used herein, the term "alkylsulfinylalkyl" refers to an alkyl group, as defined herein, substituted with an alkylsulfinyl group. Examples of unsubstituted alkylsulfinylalkyl groups are 2 to 12, 2 to 20, or 2 to 40 carbons. In some embodiments, each alkyl group can be further substituted with 1, 2, 3, or 4 substituents, as defined herein.

[0172] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can be optionally substituted with 1, 2, 3, or 4 substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituents described herein.

[0173] As used herein, the term "amidine" refers to -C(=NH)NH 2 Represents a group.

[0174] As used herein, the term “amino” refers to —N(R N1 ) 2 In the formula, each R N1 are independently H, OH, and NO 2 , N(R N2 ) 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2 , an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl, an alkaryl, a cycloalkyl, an alkylcycloalkyl, a carboxyalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any as described herein), a sulfoalkyl, an acyl (e.g., acetyl, trifluoroacetyl, or others as described herein), an alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any as described herein), a heterocyclyl (e.g., a heteroaryl), or an alkylheterocyclyl (e.g., an alkylheteroaryl), wherein these enumerated R N1 Each of the groups can be optionally substituted as defined herein for each group, or two R N1 can be combined to form a heterocyclyl or N-protecting group, N2 is independently H, alkyl, or aryl. The amino group of the present disclosure can be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R') 2 In a preferred embodiment, the amino can be -NH2 or -NHR N1 where R N1 are independently OH, NO 2 , N.H. 2 , N.R. N2 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2, alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 is H, C1-20 alkyl (e.g., C 1-6 alkyl), or C 1-10 It can be aryl.

[0175] As used herein, the term "amino acid" refers to a compound that includes a side chain, an amino group, and an acid group (e.g., -CO 2 H carboxy group or -SO 3 H sulfo group) and the amino acid is attached to the parent molecular group by a side chain, an amino group, or an acid group (e.g., a side chain). In some embodiments, the amino acid is attached to the parent molecular group by a carbonyl group, and the side chain or amino group is attached to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkylaryl, alkylheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Examples of amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolidine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid group may be optionally substituted with one, two, three, or, in the case of an amino acid group of two or more carbons, four substituents independently selected from the group consisting of: (1) C 1-6 Alkoxy, (2) C 1-6 alkylsulfinyl, (3) amino as defined herein (e.g., unsubstituted amino (i.e., —NH 2 ) or substituted amino (i.e., -N(R N1 ) 2 , where R N1 is as defined for amino), (4) C 6-10Aryl-C 1-6 Alkoxy, (5) azido, (6) halo, (7) (C 2-9 heterocyclyl)oxy, (8) hydroxy, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1-7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) -CO 2 R A’ , where R A’ (a)C 1-20 Alkyl (e.g., C 1-6 alkyl), (b) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c) C 6-10 Aryl, (d) hydrogen, (e) C 1-6 Alkyl-C 6-10 Aryl, (f) Amino-C 1-20 Alkyl, (g)-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R' is a polyethylene glycol of formula OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1-20 (h)-NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 alkyl, (15)—C(O)NR B 'R C ', in the formula, RB’ and R C’ each of which is independently (a) hydrogen, (b) C 1-6 Alkyl, (c) C 6-10 Aryl, (d) C 1-6 Alkyl-C 6-10 Aryl, (16)-SO 2 R D’ , where R D ' is (a)C 1-6 (b) C 6-10 Aryl, (c) C 1-6 Alkyl-C 6-10 Aryl, and (d) hydroxy, (17) -SO 2 NR E 'R F ', in the formula, R E’ and R F’ each of which is independently (a) hydrogen, (b) C 1-6 Alkyl, (c) C 6-10 Aryl and (d) C 1-6 Alkyl-C 6-10 Aryl, (18)-C(O)R G ', in the formula, R G ' is (a)C 1-20 Alkyl (e.g., C 1- 6 alkyl), (b) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c) C 6~10 Aryl, (d) hydrogen, (e) C 1-6 Alkyl-C 6-10 Aryl, (f) Amino-C 1-20 Alkyl, (g){ut}-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R' is H or C. 1-20 (h)-NR N1 (CH 2) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 Alkyl, (19)-NR H’ C(O)R I’ , where R H’ is (a1) hydrogen and (b1) C 1-6 alkyl, and R I’ is (a2)C 1-20 Alkyl (e.g., C 1~6 (b2) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c2) C 6-10 Aryl, (d2) hydrogen, (e2) C 1-6 Alkyl-C 6-10 Aryl, (f2) Amino-C 1-20 Alkyl, (g2)-(CH2) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R' is H or C. 1-20 Alkyl, (h 2 )-NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 Alkyl, (20)-NR J’ C(O)OR K’ , where R J’ is (a1) hydrogen and (b1) C 1-6 alkyl, and R K ' is (a2)C 1-20 Alkyl (e.g., C 1- 6 alkyl), (b2) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c2) C 6- 10 Aryl, (d2) Hydrogen, (e2) C 1-6 Alkyl-C 6-10 Aryl, (f2) Amino-C 1-20 Alkyl, (g2)-(CH2) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R' is H or C. 1-20 Alkyl, (h 2 )-NR N1 (CH) s2 (CH 2 CH 2 O)s1(CH 2 ) s3 NR N1 wherein s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), each of s2 and s3 is independently an integer from 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6(21) amidine, and (22) alkyl. In some embodiments, each of these groups can be further substituted as described herein.

[0176] As used herein, the term "aminoalkyl" refers to an alkyl group, as defined herein, substituted by an amino group, as defined herein. The alkyl and amino each may have one, two, three, or four substituents as described herein for the respective group (e.g., CO 2 R A’ , where R A’ (a)C 1-6 Alkyl, (b) C 6-10 aryl, (c) hydrogen, and (d) C 1-6 Alkyl-C 6-10 The aryl may be further substituted with an N-protecting group, such as selected from the group consisting of aryl, carboxy, and / or an N-protecting group.

[0177] As used herein, the term "aminoalkenyl" refers to an alkenyl group, as defined herein, substituted by an amino group, as defined herein. The alkenyl and amino each may have one, two, three, or four substituents, as described herein for the respective group (e.g., CO 2 R A’ , where R A’ (a)C 1-6 Alkyl, (b) C 6-10 aryl, (c) hydrogen, and (d) C 1-6 Alkyl-C 6-10 The aryl may be further substituted with an N-protecting group, such as selected from the group consisting of aryl, carboxy, and / or an N-protecting group.

[0178] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0179] The phrase "at least one" preceding a list of items with the term "and" or "or" separating any of the items modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one" does not require the selection of at least one of each of the listed items; rather, the phrase allows for a meaning including any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. As an example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers to A only, B only, or C only, any combination of A, B, and C, and / or at least one of each of A, B, and C, respectively.

[0180] When the terms "include," "have," or similar terms are used in the description or claims, such terms are intended to be inclusive in the same manner as the term "comprise" when it is used as a transitional term in a claim.

[0181] Reference to a singular element is not intended to mean "one and only one" unless otherwise specified, but rather "one or more". Masculine pronouns (e.g., his) include feminine and neuter genders (e.g., hers and its), and vice versa. The term "some" refers to one or more. Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject art, and are not referred to in connection with interpreting the description of the subject art. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure that are known or later become known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the subject art. Furthermore, any disclosures disclosed herein are not intended to be directed to the general public, regardless of whether such disclosures are expressly recited in the description above.

[0182] The term biocompatible means compatible with living cells, tissues, organs, or systems, posing little or no risk of injury, toxicity, or rejection by the immune system.

[0183] The term "biodegradable" means capable of being broken down into harmless products by the action of living organisms.

[0184] The phrase "biologically active" refers to the property of any substance that has activity in a biological system and / or organism. For example, a substance that has a biological effect on an organism when administered to the organism is considered to be biologically active. In certain embodiments, the polynucleotides of the present disclosure may be considered to be biologically active even if a portion of the polynucleotide mimics an activity that is considered to be biologically active or biologically relevant.

[0185] As used herein, the terms "carbocyclic" and "carbocyclyl" refer to optionally substituted C rings in which the rings, which may be aromatic or non-aromatic, are formed by carbon atoms. 3-12It refers to monocyclic, bicyclic, or tricyclic structures. Carbocyclic structures include cycloalkyl groups, cycloalkenyl groups, and aryl groups.

[0186] As used herein, the term “carbamoyl” refers to —C(O)—N(R N1 ) 2 In the formula, each R N1 The meaning of is found in the definition of "amino" provided herein.

[0187] As used herein, the term "carbamoylalkyl" refers to an alkyl group, as defined herein, substituted by a carbamoyl group, as defined herein. The alkyl group can be further substituted with one, two, three, or four substituents as described herein.

[0188] As used herein, the term "carbamyl" has the structure -NR N1 C(=O)OR or -OC(=O)N(R N1 ) 2 wherein each R N1 The meaning of is found in the definition of "amino" provided herein, and R is alkyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocyclyl (e.g., heteroaryl), or alkylheterocyclyl (e.g., alkylheteroaryl), as defined herein.

[0189] As used herein, the term "carbonyl" refers to a C(O) group, which also may be represented as C=O.

[0190] The term "carboxaldehyde" refers to an acyl group having the structure -C(O)H.

[0191] As used herein, the term "carboxy" refers to -CO 2 It means H.

[0192] The term "cationic lipid" refers to amphipathic lipids and their salts that have a positive hydrophilic head group, one, two, three or more hydrophobic fatty acid or fatty alkyl chains, and a connector between these two domains. An ionizable cationic lipid or a protonizable cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa and is substantially neutral at a pH above its pKa. Preferred ionizable cationic lipids are lipids that have a pKa below physiological pH, typically about 7.4. The cationic lipids of the present disclosure may also be referred to as titratable cationic lipids. The cationic lipids may be "amino lipids" that have a protonizable tertiary amine (e.g., pH titratable) head group. Some amino exemplary amino lipids may include C18 alkyl chains, each alkyl chain independently having 0-3 (e.g., 0, 1, 2, or 3) double bonds, and an ether bond, ester bond, or ketal bond between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-Bl 1).

[0193] The term "comprising" is intended to be open-ended, allowing, but not requiring, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is therefore also included and disclosed.

[0194] The term "composition" means a product comprising the specified ingredients in the specified amounts, as well as any product that results directly or indirectly from combining the specified ingredients in the specified amounts.

[0195] The term "in combination with" refers to administration of the lipid-formulated mRNA of the present disclosure together with another agent in the therapeutic methods of the present disclosure, and means that the lipid-formulated mRNA of the present disclosure and the other agent are administered sequentially or simultaneously in separate dosage forms, or simultaneously in the same dosage form.

[0196] The term "commercially available chemicals" and chemicals used in the examples described herein may be obtained from standard commercial sources, such as Acros Organics (Pittsburgh, PA), Sigma-Adrich Chemical (Milwaukee, WI), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Bron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, CA), Easman Organic Chemicals, Easton Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Frontier Scientific (Logan, UT), ICN Biomedicals, Inc. (Costa Mesa, CA), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, IL), Riedel de Haen (Hannover, Germany), Spectrence Quality Assurance (San Diego, CA), and others. Products, Inc. (New Brunswick, NJ), TCI America (Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, VA).

[0197] The phrase "compounds described in the chemical literature" can be identified through reference books and databases covering chemical compounds and chemical reactions, as known to those skilled in the art. Suitable reference books and articles detailing the synthesis of reactants useful for preparing the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York; S.R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H.O. House, "Modern Synthetic Reactions," 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif., 1972; T.L. Lichrist, "Heterocyclic Chemistry," 2nd Ed. John Wiley and Sons, New York, 1992; J. March, "Advanced Organic Chemistry: reactions, Mechanisms and Structure," 5th Ed., Wiley Interscience, New York, 1999. York, 2001; specific and similar reactants can also be identified through the index of known chemicals produced by the Chemical Abstract Service of the American Chemical Society, which is available in most public and university libraries as well as online databases (for more information, contact the American Chemical Society, Washington, DC). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis companies, where many of the standard chemical supply companies (such as those listed above) offer custom synthesis services.

[0198] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of 3 to 8 carbons and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycloheptyl, and the like. When a cycloalkyl group contains one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. Cycloalkyl groups of the present disclosure include: (1) C 1-7 Acyl (e.g., carboxaldehyde), (2) C 1-20 Alkyl (e.g., C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkylsulfinyl-C 1-6 Alkyl, Amino-C 1-6 Alkyl, azido-C 1-6 Alkyl, (Carboxaldehyde)-C 1-6 Alkyl, halo-C 1-6 Alkyl (e.g., perfluoroalkyl), hydroxy-C 1-6 Alkyl, nitro-C 1-6 Alkyl, or C 1-6 Thioalkoxy-C 1-6 (3) C 12 Alkoxy (e.g., C 1-6 alkoxy, for example, perfluoroalkoxy), (4) C 1-6 Alkylsulfinyl, (5)C 6-10 Aryl, (6) Amino, (7) C 1-6 Alkyl-C 6-10 Aryl, (8) Azide, (9) C 3-8 Cycloalkyl, (10)C 1-6 Alkyl-C 3-8 Cycloalkyl, (11) halo, (12) C 1-12 Heterocyclyl (e.g., C 1-12 Heteroaryl), (13)(C 1-12 (14) hydroxy, (15) nitro, (16) C 1-20Thioalkoxy (e.g., C 1-6 Thioalkoxy), (17)-(CH 2 ) q CO 2 R A ', where q is an integer from zero to four, and R A’ (a)C 1-6 Alkyl, (b) C 6-10 Aryl, (c) hydrogen, (d) C 1-6 Alkyl-C 6-10 aryl, (18)—(CH 2 ) q CONR B’ R C’ where q is an integer from zero to four, and where R B’ and R C’ is (a) hydrogen, (b) C 6-10 Alkyl, (c) C 6-10 Aryl, (d) C 1-6 Alkyl-C 6-10 aryl, (19)—(CH 2 ) q SO 2 R D ', where q is an integer from zero to four, and R D’ (a)C 6-10 (b) C 6-10 Aryl, (c) C 1-6 Alkyl-C 6-10 Aryl, (20)-(CH 2 ) q SO 2 NR E ' RF ', where q is an integer from zero to four, and R E’ and R F’ Each of these is (a) hydrogen, (b) C 6-10 Alkyl, (c) C 6-10 Aryl, (d) C 1-6 Alkyl-C 1-10 (21) thiol; (22) C 6-10 Aryloxy, (23)C 3-8 Cycloalkoxy, (24)C 6-10Aryl-C 1-6 Alkoxy, (25)C 1-6 Alkyl-C 1-12 Heterocyclyl (e.g., C 1-6 Alkyl-C 1-12 Heteroaryl), (26) oxo, (27) C 2-20 Alkenyl, and (28)C 2-20 In some embodiments, each of these groups can be further substituted as described herein. For example, C 1 - Alkaryl or C 1 The alkyl group of the -alkylheterocyclyl can be further substituted with oxo groups to give the respective aryloyl and (heterocyclyl)oyl substituents.

[0199] As used herein, the term "diastereomers" means stereoisomers that are not mirror images of one another and are not superimposable on each other.

[0200] The term "diacylglycerol" or "DAG" refers to a diacylglycerol consisting of two fatty acyl chains, R 1 and R 2 and R 2 , both of which have 2-30 carbons attached independently to the 1- and 2-positions of glycerol by ester bonds. The acyl groups can be saturated or have various degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18), and icosoyl (C20). In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), and R 1 and R 2 are both stearoyl (i.e., distearoyl).

[0201] The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains, R and R', both of which independently have between 2 and 30 carbons. The alkyl groups can be saturated or have varying degrees of unsaturation.

[0202] As used herein, the term "effective amount" of an agent is an amount sufficient to bring about a beneficial or desired result, e.g., a clinical result, and thus "effective amount" depends on the context in which it is applied. For example, in the context of administering an agent to treat cancer, an effective amount of an agent is an amount sufficient to achieve treatment of cancer as defined herein, e.g., compared to the response obtained without administration of the agent.

[0203] As used herein, the term "enantiomer" means each individual optically active form of a compound of the present disclosure having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90%, and more preferably at least 98%.

[0204] The term "fully encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free RNA.When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded, more preferably less than 10%, and most preferably less than 5% is degraded in a treatment that would normally degrade 100% of free nucleic acid."Fully encapsulated" also means that the nucleic acid-lipid particle is not rapidly degraded into its component parts when administered in vivo.

[0205] As used herein, the terms "halo" and "halogen" refer to a halogen selected from bromine, chlorine, iodine, or fluorine.

[0206] As used herein, the term "haloalkyl" refers to an alkyl group, as defined herein, substituted with a halogen group (i.e., F, Cl, Br, or I). Haloalkyl may be substituted with 1, 2, 3, or, in the case of alkyl groups of 2 or more carbons, 4 halogens. Haloalkyl groups include perfluoroalkyl (e.g., -CF 3 ), -CHF 2 , -CH 2 F, -CCl 3 , -CH 2 CH 2 Br, -CH 2 CH(CH 2 CH 2 Br)CH 3 , and -CHICH 3 In some embodiments, the haloalkyl group can be further substituted with 1, 2, 3, or 4 substituents described herein for an alkyl group.

[0207] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or two of the constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, the heteroalkyl group can be further substituted with one, two, three, or four substituents as described herein for the alkyl group.

[0208] As used herein, the term "hydrocarbon" refers to a group consisting solely of carbon and hydrogen atoms.

[0209] As used herein, the term "hydroxy" refers to an -OH group. In some embodiments, the hydroxy group can be further substituted with 1, 2, 3, or 4 substituents as defined herein for alkyl (e.g., O-protecting groups).

[0210] As used herein, the term "hydroxyalkenyl" refers to an alkenyl group, as defined herein, substituted with one to three hydroxy groups, provided that not more than one hydroxy group may be attached to a single carbon atom of the alkyl group, as exemplified by dihydroxypropenyl, hydroxyisopentenyl, etc. In some embodiments, the hydroxyalkenyl group can be further substituted with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0211] As used herein, the term "hydroxyalkyl" refers to an alkyl group, as defined herein, substituted with one to three hydroxy groups, provided that not more than one hydroxy group may be attached to a single carbon atom of the alkyl group, as exemplified by dihydroxypropenyl, hydroxyisopentenyl, etc. In some embodiments, the hydroxyalkyl group can be further substituted with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein for alkyl. The term "hydrate" refers to the solvent molecules being H 2 O.

[0212] As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the present disclosure. It is recognized that the compounds of the present disclosure may have one or more chiral centers and / or double bonds and thus exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present disclosure, the chemical structures depicted herein, and thus the compounds of the present disclosure, encompass all of the corresponding stereoisomers, i.e., stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallization of the compounds as chiral salt complexes, or crystallization of the compounds in chiral solvents, etc. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0213] As used herein, the term "nitro" refers to -NO 2 Represents a group.

[0214] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. This term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0215] The term "oxo" as used herein refers to =O.

[0216] As used herein, the term "stereoisomer" refers to all the different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may possess, in particular all possible stereochemical and conformational isomers, all diastereomers, enantiomers, and / or conformational isomers of a basic molecular structure. Some compounds of the present disclosure may exist in different tautomeric forms, all of the latter being included within the scope of the present disclosure.

[0217] As used herein, the term "sulfonyl" refers to -S(O) 2 - represents a group.

[0218] The term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[0219] The term "conserved" refers to nucleotides or amino acid residues, respectively, of a polynucleotide or polypeptide sequence that occur unchanged in the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is one that is more conserved between related sequences than a nucleotide or amino acid that appears elsewhere in the sequences.

[0220] The term "circular" refers to the presence of a continuous ring. A circular molecule does not have to be circular, but only joins to form an uninterrupted chain of subunits. A circular molecule, such as an mRNA of the present disclosure, may be a single unit or a multimer, or may contain one or more components of a complex or higher order structure.

[0221] The term "cytotoxicity" refers to killing or causing an injurious, toxic, or lethal effect on a cell (e.g., a mammalian cell (e.g., a human cell)), bacteria, virus, fungus, protozoan, parasite, prion, or a combination thereof.

[0222] The term "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload.

[0223] The term "delivery agent" refers to any substance that facilitates, at least in part, the in vivo delivery of a polynucleotide to a target cell.

[0224] The term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), (3) translation of the RNA into a polypeptide or protein, and (4) post-translational modification of the polypeptide or protein.

[0225] The term "characteristic" refers to a characteristic, property, or distinctive element.

[0226] As used herein, the term "fragment" refers to a portion. For example, a fragment of a protein may include a polypeptide obtained by digesting a full-length protein isolated from a cultured cell.

[0227] The term "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0228] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic group(s).Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane, and l,2-dialkyl-3-aminopropane.

[0229] The term "lipid" refers to organic compounds that contain esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes, (2) "complex lipids," which include phospholipids and glycolipids, and (3) "derived lipids," such as steroids.

[0230] The term "lipid delivery vehicle" refers to a lipid formulation that can be used to deliver therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, etc.). The lipid delivery vehicle can be a nucleic acid-lipid particle that can be formed from cationic lipids, non-cationic lipids (e.g., phospholipids), conjugated lipids that prevent particle aggregation (e.g., PEG lipids), and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) is encapsulated in the lipid portion of the particle, which can protect it from enzymatic degradation.

[0231] The term "lipid encapsulation" refers to lipid particles that provide a therapeutic nucleic acid, such as an mRNA, with complete encapsulation, partial encapsulation, or both. In preferred embodiments, the nucleic acid (e.g., the mRNA) is completely encapsulated within the lipid particle.

[0232] The term "amphipathic lipid" or "amphiphilic lipid" refers to a material in which the hydrophobic portion of the lipid material orients toward the hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups such as carbohydrate, phosphate, carboxylic acid, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0233] The term "linker" or "linking moiety" refers to a group of atoms, e.g., 10-100 atoms, which may consist of atoms or groups, such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be of sufficient length so as not to interfere with incorporation into the amino acid sequence. Examples of chemical groups that may be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkyl, heteroalkyl, aryl, or heterocyclyl, each of which may be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties in the linker, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or photolysis, as well as ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0234] The term mammal means a human or other mammal, or a human.

[0235] The term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein or polypeptide of interest.

[0236] The term "modification" refers to a change in the state or structure of a molecule of the present disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. In one embodiment, a nucleic acid active component is modified by the introduction of non-natural nucleosides and / or nucleotides, for example, as they relate to the natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, may differ from the chemical structure of A, C, G, U ribonucleotides, but are not considered "modified."

[0237] The term "naturally occurring" means existing in nature without artificial assistance.

[0238] The term "non-human vertebrate" includes all vertebrates except Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals, such as alpacas, bantengs, bisons, camels, cats, cows, deer, dogs, donkeys, gayal, goats, guinea pigs, horses, llamas, mules, pigs, guinea pigs, rabbits, reindeer, sheep, buffalo, and yaks.

[0239] The term "nucleotide" refers to natural (standard) and modified bases known in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally comprise a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified in the sugar, phosphate, and / or base moieties (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and others; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92 / 07065; Usman et al., International PCT Publication No. WO 93 / 15187; Uhlman & Peyman, supra, all of which are incorporated herein by reference). There are several examples of modified nucleobases known in the art, as summarized in Limbach, et al., Nucleic Acids Res. 22:2183, 1994. Non-limiting examples of base modifications that can be introduced into nucleic acid molecules include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, etc. (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). In this embodiment, "modified base" refers to a nucleotide base other than adenine, guanine, cytosine, thymine, and uracil at the 1' position, or their equivalents.

[0240] The phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, and the like.

[0241] The term "patient" refers to a subject seeking or needing treatment, a subject in need of treatment, a subject receiving treatment, or a subject to be treated, or a subject receiving care from a trained professional for a particular disease or condition.

[0242] The phrase "optionally substituted X" (e.g., optionally substituted alkyl) is intended to be equivalent to "X, where X is optionally substituted" (e.g., "alkyl, where the alkyl is optionally substituted"). It is not intended to imply that the feature "X" (e.g., alkyl) itself is optional.

[0243] As used herein, the phrase "pharmacologically acceptable" is used to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0244] As used herein, the phrase "pharmaceutical acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle that can suspend or dissolve active compounds), and has the properties of being substantially non-toxic and non-inflammatory in patients.Excipients may include, for example, anti-adhesive agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavors, flavorings, glidants (glidants), lubricants, preservatives, printing inks, absorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0245] The phrase "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, tetrahydrofuran ... Salts include, for example, salts of sodium, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods.Generally, these salts can be prepared by reacting the free acid form or free base form of these compounds with stoichiometric amount of suitable base or acid in water or organic solvent, or in the mixture of the two, generally non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0246] The term "pharmacokinetics" refers to any one or more properties of a molecule or compound as it relates to determining the fate of a substance administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism, and excretion. This is commonly referred to as ADME, which is as follows: (A) absorption is the process of a substance entering the blood circulation; (D) distribution is the dispersion or seeding of a substance throughout the fluids and tissues of the body; (M) metabolism (or biotransformation) is the irreversible conversion of a parent compound to daughter metabolites; and (E) excretion (or elimination) refers to the removal of a substance from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

[0247] As used herein, the term "pharmaceutical acceptable solvate" refers to a compound of the present disclosure in which molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, a solvate may be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (e.g., monohydrate, dihydrate, and trihydrate), N-methylpyrrolidinone (NMP), dimethylsulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate."

[0248] The term "physicochemical" means or relates to physical and / or chemical properties.

[0249] The term "phosphate" is used in its ordinary sense as understood by those of skill in the art and includes its protonated form, e.g., [ka] and [ka] Includes. As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used in their ordinary sense as understood by those of skill in the art and include the protonated forms.

[0250] The term "phosphorothioate" refers to a compound of the general formula: [ka] , its protonated form, e.g. [ka] and its tautomers, such as [ka] and [ka] Refers to...

[0251] The term "prevention" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition, and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0252] The term "protein of interest" or "desired protein" includes those provided herein, as well as fragments, mutants, variations, and modifications thereof.

[0253] The terms "purify," "purified," and "purification" mean to make substantially pure or substantially free of undesirable components, material imperfections, admixtures, or defects.

[0254] The term "RNA" refers to a molecule that contains at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material to the end(s) of an interfering RNA or internally, for example, at one or more nucleotides of an RNA. Nucleotides in the RNA molecules of the present disclosure can also include non-naturally occurring nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of naturally occurring RNA. As used herein, the terms "ribonucleic acid" and "RNA" refer to molecules that contain at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and polyvalent RNA.

[0255] The term "sample" or "biological sample" refers to a subset of its tissues, cells, or component parts (e.g., bodily fluids, including but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). Samples may further include homogenates, lysates, or extracts prepared from whole organisms, or a subset of its tissues, cells, or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph, external sections of the skin, respiratory, intestinal, and reproductive tracts, tears, saliva, milk, blood cells, tumors, organs. Samples may further refer to media, such as nutrient broths or gels, which may contain cellular components, such as proteins or nucleic acid molecules.

[0256] The terms "significant" or "significantly" are used synonymously with the term "substantially."

[0257] The phrase "single unit dose" is a dose of any therapeutic agent administered in one dose / single route / single point of contact, i.e., a single administration event.

[0258] The term "siRNA" or small interfering RNA, sometimes known as short interfering RNA or silencing RNA, typically refers to a class of double-stranded RNA non-coding RNA molecules, 18-27 base pairs in length, similar to miRNAs, that operate within the RNA interference (RNAi) pathway, which interferes with the expression of specific genes that have complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.

[0259] The term solvate refers to the physical association of the compound of the present disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonds, including hydrogen bonds. In certain cases, the solvate has the ability to be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" includes both solution-phase and isolable solvates. Non-limiting examples of suitable solvates include ethanolate, methanolate, etc.

[0260] The term "split dose" refers to the division of a single unit dose or total daily dose into two or more doses.

[0261] The term "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and, preferably, is capable of being formulated into an efficacious therapeutic agent.

[0262] The terms "stabilize," "stabilized," and "stabilized region" mean to make stable or to become stable.

[0263] The term "substituted" means substitution with a specified group other than hydrogen, or, for example, with one or more groups, moieties, or radicals, each of which is independently selected and can be the same or different.

[0264] The term "substantially" refers to a qualitative condition that indicates the extent or degree of the total or near total of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0265] The phrase "substantially equal" refers to the time difference between doses, the term meaning ±2%.

[0266] The phrase "substantially simultaneously" refers to multiple doses, the term meaning within 2 seconds.

[0267] The phrase "suffering from" relates to an individual "suffering from" a disease, disorder, and / or condition having been diagnosed with or exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0268] The term "susceptibility" refers to an individual who is "susceptible" to a disease and / or condition who has not been diagnosed with the disease, disorder, and / or condition and / or does not show symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the onset of the disease, disorder, and / or condition, (2) a genetic polymorphism associated with the onset of the disease, disorder, and / or condition, (3) an increase and / or decrease in expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition, (4) habits and / or lifestyle associated with the onset of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, and (6) exposure to and / or infection with a microorganism associated with the onset of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who has a susceptibility to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0269] The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of a polynucleotide or polypeptide or other molecule of the disclosure may be chemical synthesis or enzymatic synthesis.

[0270] The term "target cell" refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0271] The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0272] The term "therapeutically effective amount" means an amount of an agent (e.g., a nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0273] The term "therapeutically effective outcome" means an outcome that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition.

[0274] The term "total daily dose" is the amount given or prescribed for a 24-hour period. It may be administered as a single unit dose.

[0275] The term "treatment" refers to the partial or complete alleviation, palliation, amelioration, mitigation, delay in onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treatment" of cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0276] The term "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified may, but does not necessarily, refer to a wild-type or naturally occurring biomolecule. A molecule may undergo a series of modifications, whereby each modified molecule may serve as an "unmodified" starting molecule for subsequent modifications.

[0277] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are intended in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0278] The compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the simultaneous migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in heterocyclic ring systems such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0279] The compounds of the present disclosure also include all isotopes of atoms occurring in intermediate or final compounds. "Isotopes" refers to atoms that have the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0280] The compounds and salts of the disclosure can be prepared in combination with a solvent or water molecules to form solvates and hydrates by routine methods.

[0281] The term "half-life" is the time required for a quantity, such as the concentration or activity of a nucleic acid or protein, to decrease to half of its value as measured at the beginning of a period of time.

[0282] The term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.

[0283] The term "in vivo" refers to an event that takes place within a living organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0284] The term "monomer" refers to a single unit, e.g., a single nucleic acid that can combine with another molecule of the same or different type to form an oligomer. In some embodiments, the monomer may be a non-locked nucleic acid, i.e., a UNA monomer.

[0285] The term "neutral lipid" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0286] The term "non-cationic lipid" means an amphipathic lipid or a neutral lipid or an anionic lipid, as described herein.

[0287] The term "subject" or "patient" refers to any living organism to which a composition according to the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0288] The term "translatable" may be used interchangeably with the term "expressible" and refers to the ability of a polynucleotide or a portion thereof to be converted into a polypeptide by a host cell. As understood in the art, translation is the process by which ribosomes in the cytoplasm of a cell generate a polypeptide. In translation, messenger RNA (mRNA) is decoded by tRNA in the ribosomal complex to produce a specific amino acid chain or polypeptide. Furthermore, when used herein with respect to an oligomer, the term "translatable" means that at least a portion of the oligomer, such as the coding region (also known as the coding sequence or CDS) of the oligomer sequence, has the ability to be converted into a protein or a fragment thereof.

[0289] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means an outcome that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition.

[0290] The term "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient may generally be equal to the dose of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dose, including, but not limited to, one-half or one-third of such a dose.

[0291] "Control" or "control experiment" is used according to its plain and ordinary meaning to refer to an experiment in which experimental subjects or agents are treated as in a parallel experiment, except for the omission of an experimental procedure, agent, or variable. In some cases, a control is used as a standard of comparison in evaluating the effect of an experiment.

[0292] While the present disclosure has been described in connection with certain specific embodiments and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may vary substantially without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications, and equivalents. In particular, the present disclosure includes any combination of the features, terms, or elements of the various exemplary components and examples.

[0293] Embodiment Embodiment 1. A lipid composition containing a nucleic acid, the lipid composition comprising a peptide-lipid conjugate. Embodiment 2. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of about 4 to about 52 amino acids. Embodiment 3. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is about 8 to about 50 amino acids in length. Embodiment 4. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 44 amino acids in length. Embodiment 5. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 40 amino acids in length. Embodiment 6. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 36 amino acids in length. Embodiment 7. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 32 amino acids in length. Embodiment 8. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 28 amino acids in length. Embodiment 9. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is from about 8 to about 24 amino acids in length. Embodiment 10. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate is about 8 to about 20 amino acids in length. Embodiment 11. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of 12 amino acids. Embodiment 12. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of 16 amino acids. Embodiment 13. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of 20 amino acids. Embodiment 14. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of 24 amino acids. Embodiment 15. The lipid composition of embodiment 1, wherein the peptide of the peptide-lipid conjugate consists of 32 amino acids. Embodiment 16. The lipid composition of any one of the preceding embodiments, wherein at least about 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline. Embodiment 17. The lipid composition of embodiment 15, wherein about 14% to about 60% of the amino acids in the peptide are proline. Embodiment 18. The lipid composition of embodiment 15, wherein about 20% to about 60% of the amino acids in the peptide are proline. Embodiment 19. The lipid composition of embodiment 15, wherein about 25% to about 60% of the amino acids in the peptide are proline. Embodiment 20. The lipid composition of any one of the preceding embodiments, wherein at least about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have a hydrophilic side chain. Embodiment 21. The lipid composition according to any of embodiments 1 to 19, wherein about 35% to about 85% of the amino acids in the peptide have a hydrophilic side chain. Embodiment 22. The lipid composition according to any one of embodiments 1 to 19, wherein about 35% to about 80% of the amino acids in the peptide have a hydrophilic side chain. Embodiment 23. The lipid composition according to any one of embodiments 1 to 19, wherein about 40% to about 75% of the amino acids in the peptide have a hydrophilic side chain. Embodiment 24. The lipid composition according to any one of embodiments 1 to 19, wherein about 45% to about 85% of the amino acids in the peptide have a hydrophilic side chain. Embodiment 25. The lipid composition according to any one of embodiments 1 to 19, wherein about 50% to about 75% of the amino acids in the peptide have a hydrophilic side chain. Embodiment 26. The amino acid having a hydrophilic side chain is selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, serine, OC 1-6 Alkylserine, Threonine, and OC 1-6 2. The lipid composition of any one of the preceding embodiments, wherein the lipid is selected from alkyl threonines. Embodiment 27. The lipid composition of any one of the preceding embodiments, wherein less than about 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine. Embodiment 28. The lipid composition of embodiment 27, wherein less than about 30% of the amino acids in the peptide are glycine. Embodiment 29. The lipid composition of embodiment 27, wherein less than about 20% of the amino acids in the peptide are glycine. Embodiment 30. The lipid composition of embodiment 27, wherein less than about 10% of the amino acids in the peptide are glycine. Embodiment 31. The lipid composition of embodiment 27, wherein less than about 5% of the amino acids in the peptide are glycine. Embodiment 32. The lipid composition of any one of the preceding embodiments, wherein the peptide has no glycines. Embodiment 33. The lipid composition of any one of the preceding embodiments, wherein the lipid composition is selected from lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions. Embodiment 34. The lipid composition according to embodiment 33, wherein the liposomes are selected from cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. Embodiment 35. The lipid composition of embodiment 33, wherein the lipid composition is a lipid nanoparticle. Embodiment 36. The lipid composition of embodiment 35, wherein the lipid nanoparticles have a size of less than about 200 nm. Embodiment 37. The lipid composition of any one of the preceding embodiments, wherein the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol. Embodiment 38. The lipid composition of any one of the preceding embodiments, wherein the peptide-lipid conjugate comprises from about 0.1 mol % to about 10 mol % of all lipids in the lipid composition. Embodiment 39. The lipid composition of embodiment 38, wherein the peptide-lipid conjugate comprises from about 0.5 mol % to about 5 mol % of all lipids in the lipid composition. Embodiment 40. The lipid composition of embodiment 38, wherein the peptide-lipid conjugate comprises from about 1.0 mol % to about 3 mol % of all lipids in the lipid composition. Embodiment 41. The lipid composition of any one of the preceding embodiments, wherein the lipid composition encapsulates a nucleic acid. Embodiment 42. The lipid composition of any one of embodiments 1 to 40, wherein the lipid composition is complexed to a nucleic acid. Embodiment 43. The lipid composition of any one of the preceding embodiments, wherein the lipid of the peptide-lipid conjugate is conjugated to the peptide via a linker. Embodiment 44. The linker is an amide (-C(O)NH-), an amino (-NR N -), wherein R N But, H, C 1-6 Alkyl, carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH 2CH 2 C(O)-), succinamidyl (-NHC(O)CH 2 CH 2 44. The lipid composition of embodiment 43, wherein the lipid is selected from the group consisting of C(O)NH-), ether, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), and sulfonate ester. Embodiment 45. The lipid of the peptide-lipid conjugate is didecyloxypropyl (C 10 ), dilauryloxypropyl (C 12 ), Dimyristyloxypropyl (C 14 ), dipalmityloxypropyl (C 16 ), or distearyloxypropyl (C 18 ), 1,2-dipalmityloxypropyl-3-amine (DOMG), 1,2-dimyristyloxypropylamine (DMG), 1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine (DLPE), dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), and cholesterol or a cholesterol derivative. Embodiment 46. The peptide is conjugated at its C-terminus to a lipid of a peptide-lipid conjugate, and the amino group at the N-terminus of the peptide is substituted with one or two C 1-6 13. The lipid composition of any one of the preceding embodiments, wherein the lipid composition is substituted with an alkyl or amide group. Embodiment 47. The peptide is conjugated at its N-terminus to a lipid of a peptide-lipid conjugate, and the amino acid at the C-terminus of the peptide is alkylated to C 1-6 The lipid composition according to any one of embodiments 1 to 45, which forms an alkyl ester or is amidated. Embodiment 48. 5-Carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium propane-(DODAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DSDMA), Di-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyrityloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium monium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-oc-tadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA) , 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and 2,The lipid composition according to any one of embodiments 37 to 47, further comprising one or more cationic lipids selected from 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (DLin-K-XTC2-DMA). Embodiment 49. The following [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The lipid composition according to any one of embodiments 37 to 47, further comprising an ionic cationic lipid selected from: Embodiment 50. The lipid composition according to any one of embodiments 1 to 49, further comprising one or more helper lipids. Embodiment 51. A lipid composition comprising a peptide-lipid conjugate and a nucleic acid, a. the peptide of the peptide-lipid conjugate consists of about 4 to about 52 amino acids; b. at least about 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline; c. about 28% to about 80% of the amino acids in the peptide of the peptide-lipid conjugate have hydrophilic side chains; d. less than about 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine; e. the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol; f. A lipid composition, wherein the peptide-lipid conjugate comprises from about 0.1 mol % to about 10 mol % of all lipids in the lipid composition. Embodiment 52. The lipid composition of any one of embodiments 1 to 51, wherein the nucleic acid is selected from siRNA, antisense oligonucleotide, UNA oligomer, mRNA, microRNA, and DNA. Embodiment 53. The lipid composition of embodiment 52, wherein the nucleic acid is mRNA. Embodiment 54. The lipid composition of embodiment 53, wherein the mRNA is a self-replicating mRNA. Embodiment 55. The lipid composition of embodiment 52, wherein the nucleic acid is an siRNA. Embodiment 56. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 12 amino acids. Embodiment 57. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 16 amino acids. Embodiment 58. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 20 amino acids. Embodiment 59. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 24 amino acids. Embodiment 60. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 28 amino acids. Embodiment 61. The lipid composition according to any one of embodiments 51 to 55, wherein the peptide consists of 32 amino acids. Embodiment 62. A method of treating a disease in a subject in need thereof comprising administering to the subject a lipid composition according to any one of the preceding embodiments. Embodiment 63. A method of expressing a protein or polypeptide of interest in a cell, comprising contacting the cell with a lipid composition according to embodiment 53 or 54. Embodiment 64. A vaccine comprising the lipid composition of embodiment 53 or 54. Embodiment 65. A method of eliciting an immune response in a subject comprising administering to the subject the vaccine of embodiment 64. Embodiment 66. A method of inhibiting expression of a gene or messenger RNA of interest in a cell, comprising contacting the cell with the lipid composition of embodiment 55. Embodiment 67. A method of expressing a protein or polypeptide of interest in a subject lacking said protein or polypeptide, comprising administering to the subject the lipid composition of embodiment 53, wherein the mRNA encodes the protein or polypeptide of interest. Embodiment 68. A method of editing a gene in a cell comprising contacting the cell with the lipid composition of embodiment 53, wherein the mRNA encodes a gene-editing enzyme.

[0046] Embodiment 69. The method of embodiment 68, wherein the gene editing enzyme is a TALEN enzyme. Embodiment 70. A method of delivering a nucleic acid to a cell comprising contacting the cell with a lipid composition according to any one of embodiments 1 to 61. Embodiment 71. A method for preventing a disease in a subject, comprising administering to the subject a lipid composition according to any one of embodiments 1 to 61. Embodiment 72. The method of any one of embodiments 62, 65, 67, or 71, wherein administration is intravenous or intramuscular. Embodiment 73. A method of imaging a cell comprising contacting the cell with a lipid composition according to any one of embodiments 1 to 61, wherein the nucleic acid encodes a detectable protein. EMBODIMENT 74. i) contacting a lipid nucleic acid with a peptide-lipid conjugate; ii) enabling the peptide-lipid conjugate to encapsulate a nucleic acid. EXAMPLES

[0294] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0295] Example 1: Synthesis of peptides and examples of peptide-lipid conjugates Peptide synthesis Generally, peptides were synthesized on a peptide synthesizer using standard N-(9-fluorenylmethoxycarbonyloxy) (Fmoc) protecting group (B) chemistry and purified on a C18 column using HPLC. Briefly, peptide synthesis was carried out in a linear fashion on a Prelude X peptide synthesizer (Protein Technologies, Inc., Tucson, AZ) following the solid-phase peptide synthesis protocol using Fmoc-protected amino acids, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(Me)-OH, FmocThr(Me)-OH as building block reagents and N,N-dimethylformamide, acetonitrile, diethyl ether, and dichloromethane as the solvents of choice for the various steps. First, Fmoc-Pro-OH was loaded onto 2-ClTrityl resin (0.6 equivalents relative to resin, 4 equivalents relative to N,N-diisopropylethylamine (DIEA)). Fmoc was then deprotected using 20% ​​piperidine (2×5 min). Subsequently, 7.5 equivalents of the desired Fmoc-AA, HCT, were coupled as the activator and 15 equivalents of NNM as the base. A double coupling approach was used for 25 and 20 min to ensure complete coupling. The Fmoc deprotection and double coupling steps were repeated for all amino acids and until the desired peptide was synthesized. Each peptide on the resin was dried and cleaved from the resin using a cocktail of 90% TFA, 5% thioanisole, 2.5% H2O, 1.5% ethanedithiol, and 1% phenol by volume at ambient temperature for 2 h. Further, each peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Jupiter 10u Proteo column (Phenomenex, Torrance, CA) with a size of 250 × 21.2 mm. A mobile phase of solvent A of 0.1% TFA in H2O and solvent B of 0.1% TFA in 80% acetonitrile was used with a gradient of 18% to 38% mobile phase B within 20 min. A flow rate of 15 ml / min and a UV detection wavelength of 214 nm were used.The major product containing fractions were analyzed, pooled and the solvent removed to give the pure peptide.

[0296] To form peptide-lipid conjugates from the peptides, each peptide was coupled at the N-terminal amine with (R)-2,3-bis(tetradecanoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate (compound 3 below) to obtain the final DMG-SA-peptide conjugates. Briefly, the linear peptide obtained above and DMG-SA-NHS (N-hydroxysuccinimide) (formula 1:1.2) are dissolved in DMF (dimethylformamide) overnight in the presence of 2 equivalents of DIEA. The formed products (DMG-SA-peptides) were then precipitated in cold ether. These conjugates were further purified on a C8 column and lyophilized at -80 °C on a Labconco freeze dryer (Kansas City, MO) without any additional additives to obtain the pure products as white powders. The final yields ranged from about 60 to 80%. The coupling reaction and conjugated lipid described in this example are chosen to provide proof of concept for the conjugated peptides of the present disclosure, and those skilled in the art will recognize other suitable coupling reactions and lipids known in the art for conjugation with the peptides of the present disclosure.In addition, methods for coupling a peptide at its C-terminus or at one of its amino acid side chains are well known in the art.

[0297] Examples of peptides generated in this study are listed in Table 1 below. [Table 1]

[0298] Synthesis of DMG-peptide conjugates Exemplary peptide-lipid conjugates were made using the peptides described herein conjugated to the exemplary lipid compound (R)-2,3-bis(tetradecanoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate (Group 3) according to synthetic scheme 1 described herein.

[0299] Scheme 1 Synthesis of DMG-peptide conjugates [ka]

[0300] i) (R)-4-(2,3-bis(tetradecanoyloxy)propoxy)-4-oxobutanoic acid (compound 2 in Scheme 1) Succinic anhydride (670 mg, 6.6 mmol) and N,N-dimethylaminopyridine (DMAP, 1.0 g, 8.3 mmol) were added to a solution of (S)-3-hydroxypropane-1,2-diylditetradecanoate (compound 1 of Scheme 1, 2.05 g, 4 mmol) in 40 mL of dichloromethane at room temperature. The mixture was stirred at ambient temperature for 16-18 h. An aliquot of 1 M aqueous hydrochloric acid (8.5 mL) was added to quench the reaction. The mixture was diluted with 20 mL of water and the organic layer was separated. The aqueous layer was extracted with another 40 mL of dichloromethane and the combined organic solution was washed with 1 M aqueous HCl (1 × 100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure on a rotary evaporator. The resulting semisolid was dried under high vacuum over phosphorus pentoxide to give 2.4 g of product as a white solid. m / z 612.46 (calculated) MH 611.7 (observed).

[0301] ii. (R)-2,3-bis(tetradecanoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate (compound 3 in Scheme 1) To a mixture of (R)-4-(2,3-bis(tetradecanoyloxy)propoxy)-4-oxobutanoic acid (8.8 g, 28.7 mmol), triethylamine (2.9 g, 19.6 mmol), and 80 mg of DMAP in 160 mL of dichloromethane, succinimidyl carbonate (5.04 g, 19.6 mmol) was added and the mixture was stirred at room temperature for 16 h. The reaction was quenched by adding 2 equivalents of glacial acetic acid. The mixture was diluted with another 100 mL of DCM and washed with ice-cold water (2×300 mL) followed by brine (1×300 mL). The organic phase was separated, dried (anhydrous sodium sulfate), and the solvent was removed under reduced pressure. The residue was purified on an 80 g Teledyne ISCO silica gel column using a gradient of dichloromethane:ethyl acetate. Fractions eluted with 10-12% ethyl acetate were pooled and concentrated under reduced pressure to give 9 g of product as a white solid, m / z 709.5 (calculated) M+Na 732.2 (observed).

[0302] iii) DMG-SA-(peptide) peptide synthesis (exemplified by compound 4 in Scheme 1) Each synthetic peptide described in this example was coupled at the N-terminal amine with (R)-2,3-bis(tetradecanoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate to give the final DMG-SA-(peptide) conjugates, which were further purified on a C8 column and lyophilized to give the pure products as white powders.

[0303] Example 2: Protocol for lipid nanoparticle preparation The peptide-lipid conjugates of the present disclosure were tested in nucleic acid-lipid formulations. Lipid nanoparticles (LNPs) encapsulating FVII siRNA or human erythropoietin (hEPO) mRNA were prepared by mixing an ethanolic solution of lipids with an aqueous solution of RNA according to the method described in Ramaswamy et al. (Proc. Natl. Acad. Sci. US A. 2017 Mar 7; 114(10): E1941-E1950). Briefly, lipid excipients (ionizable lipids, DSPC, cholesterol, and PEG2000-DMG or peptide-lipid conjugates of the present disclosure) are dissolved in ethanol at a specific molar ratio. An aqueous solution of RNA is prepared in citrate buffer at pH 3-4. The lipid mixture is then combined with the RNA solution at a flow ratio (V / V) of 1:3 using a Nanoassemblr microfluidic system (Precision NanoSystems, Vancouver, BC, Canada). The nanoparticles thus formed are purified by a tangential flow filtration (TFF) process. The concentration of the resulting formulation is then adjusted to the final target RNA concentration using 100,000 MWCO Amicon Ultra centrifuge tubes (Millipore Sigma), followed by filtration through a 0.2 μm PES sterilizing grade filter. After filtration, the bulk formulation is aseptically filled into sterile Eppendorf tubes and frozen at -70±10°C. Analytical characterization of the lipid nanoparticles includes particle size and polydispersity measurements using dynamic light scattering (ZEN3600, Malvern Instruments), RNA content, and encapsulation efficiency by fluorescence assay using RiboGreen RNA reagent (Thermo Fisher Scientific).

[0304] Example 3: Protocol for evaluating Factor VII knockdown Lipid formulations containing FVII siRNA, as described further below, were evaluated for knockdown activity using the protocol of this example. For FVII evaluation, 7-8 week old female Balb / C mice were purchased from Charles River Laboratories (Hollister, CA). Mice were kept in a pathogen-free environment and all procedures involving mice were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee (IACUC). Lipid nanoparticles containing factor VII siRNA were administered intravenously at a dose volume of 10 mL / kg and two dose levels (0.03 mg / kg and 0.01 mg / kg). After 48 hours, mice were anesthetized with isoflurane and bled retro-orbitally into Microtainer® tubes coated with 0.109 M sodium citrate buffer (BD Biosciences, San Diego, CA) and processed to plasma. Plasma specimens were either tested immediately for factor VII levels or stored at -80°C for later analysis. Measurement of FVII protein in plasma was determined using a colorimetric Biophen VII assay kit (Aniara Diagnostica, USA). Absorbance was measured at 405 nm and a calibration curve was generated using serially diluted control plasma to determine levels of factor VII in plasma from treated animals compared to saline-treated control animals.

[0305] Example 4: Protocol for assessing hEPO mRNA expression The following hEPO mRNA-containing lipid formulations were evaluated for their ability to express hEPO in vivo according to the protocol of this example. All animal experiments were performed using an institutionally approved protocol (IACUC). For this protocol, female Balb / c mice at least 6-8 weeks old were purchased from Charles River Laboratory. Mice were intravenously injected via the tail vein with hEPO-LNPs with one of two dose levels of hEPO (0.1 and 0.03 mg / kg). After 6 hours, blood was collected in serum separator tubes and serum was isolated by centrifugation. Serum hEPO levels were then measured using an ELISA assay (Human Erythropoietin Quantikine IVD ELISA Kit, R&D Systems, Minneapolis, MN, USA).

[0306] Example 5: Biodistribution and Immunostaining Protocol Studies evaluating the biodistribution and immunostaining of the formulations described herein were performed according to the protocol described in this example. In this protocol, transgenic and floxed tdTomato mice were used. These mice were engineered to have a gene encoding the tdTomato fluorescent reporter protein, but also contain a CRE-based stop cassette (i.e., a floxed cassette) that prevents full transcription of the tdTomato gene in the absence of a protein called CRE recombinase (CRE). Floxed tdTomato mice are additionally deficient in the CRE gene.

[0307] A total of six floxed tdTomato mice were divided into three groups of two mice each. The control group was injected with PBS, and the remaining two groups were injected with LNP formulations containing CRE-tdTomato mRNA. The LNP formulations included either PEG-DMG or peptide 7. One mouse from each group received an intravenous (IV) injection, and the other mouse received an intramuscular (IM) injection. Animals were administered 1 mg / kg of mRNA and a volume of 10 mL / kg. 72 hours after injection, mice were euthanized. For mice administered by IV injection, organs were removed, including liver, spleen, lungs, kidneys, and heart. For mice administered by IM injection, injection sites were removed, including left rectus femoris, right rectus femoris, liver, and spleen. Organs were fixed in 10% neutral buffered formalin, embedded into paraffin blocks, and cut into 5 μm sections. Each section was stained for secondary detection by immunohistochemistry using tdTomato antibody. Sections were then incubated with a 1:300 dilution of biotin-labeled anti-rabbit (ab6801) and stained using streptavidin-horseradish peroxidase (HRP) (20774, Millipore) and 3,3'-diaminobenzidine (DAB) substrate (SK-4100, Vector Laboratories). Images of samples were collected using a confocal immunofluorescence microscope.

[0308] The extent to which treatment of mice transfected with the CRE mRNA lipid formulation was successful was indicated by expression of tdTomato protein, thus allowing the mice to produce the CRE protein that removes the floxed cassette to allow expression of the tdTomato protein. As shown in Figure 3, LNP formulations containing the peptides described herein can efficiently deliver mRNA to mouse organs.

[0309] Example 6: Exemplary lipid nanoparticle formulations Lipid nanoparticle formulations encapsulating either FVII siRNA or hEPO mRNA were prepared as described in the protocol of Example 2 above. These lipid nanoparticle formulations included an ionic cationic lipid (Cat), a helper lipid (distearoylphosphatidylcholine, DSPC), cholesterol (Chol), and either a lipid-peptide conjugate or a PEG-lipid conjugate. The ionic cationic lipids used in these formulations were selected to provide a common lipid that can serve as a basis for comparison, but one skilled in the art will recognize that the lipid-peptide conjugates of the present disclosure can be combined with any cationic lipid suitable for use in lipid nanoparticle formulations for delivery of active agents such as nucleic acids. The ionic cationic lipids used in these formulations have the following structure: [ka] has.

[0310] Exemplary lipid nanoparticle formulations were prepared and characterized as described in Example 2, and details of each formulation, along with the resulting properties, are provided in Table 2 below. In this table, "N / P" refers to the ratio of cationic amino groups from the ionizable cationic lipid to the anionic phosphate backbone groups of the encapsulated nucleic acid. The results show that the peptide-lipid conjugates of the present disclosure are successfully integrated into lipid nanoparticle formulations with good particle size, polydispersity, and percent encapsulation of nucleic acid. [Table 2]

[0311] Example 7: EPO Expression In Vivo Each of the peptide-lipid conjugates was evaluated for its effectiveness in delivering hEPO mRNA for in vivo expression according to the protocol outlined in Example 4 at mRNA concentrations of 0.1 and 0.03 mg / kg. The PEG2000-DMG formulation was also tested at two different mole percentages of the lipid portion of the composition, 1% and 1.5%. The results of this study are shown in Figure 1. At the 0.1 mg / kg level, the peptide 2 and peptide 5 formulations are comparable to the PEG2000-DMG formulation. The peptide 6 and peptide 7 show significantly higher EPO expression than the PEG2000-DMG formulation, while the peptide 3 and peptide 8 formulations show expression levels that are much better than the PEG2000-DMG formulation. These results indicate that the peptide-lipid conjugates of the present disclosure are at least a suitable alternative to the use of PEG conjugates in lipid nanoparticles, and in some cases are much better at enhancing protein expression levels of the mRNA delivered in vivo.

[0312] Example 8: FVII knockdown in vivo The peptide-lipid conjugates were further evaluated for efficacy in knocking down factor VII (FVII knockdown) by formulating lipid nanoparticles as described above that encapsulate targeted siRNA to knock down FVII. These formulations were tested at FVII siRNA dose levels of 0.01 mg / kg and 0.03 mg / kg. Comparative formulations, otherwise identical to the lipid structure, but using either 1.0% or 1.5% PEG2000-DMG, as well as a negative control of phosphate-buffered saline (PBS), were also tested. Results normalized to PBS-expressed FVII expression levels are provided in FIG. 2. It can be seen that peptide 2 shows expression levels comparable to the 1% PEG-DMG formulation. Peptides 3, 5, 6, 7, and 8 all showed better knockdown activity than the 1% PEG-DMG formulation and comparable to the 1.5% PEG-DMG formulation. Peptide 7 showed particularly improved knockdown at the 0.03 mg / kg dose level compared to the 1.5% PEG-DMG formulation. As such, the peptide-lipid conjugates of the present disclosure are at least a suitable alternative to the use of PEG conjugates in lipid nanoparticles, and in some cases demonstrate far superior enhanced delivery and knockdown activity in vivo.

[0313] Example 9: Further peptide-lipid conjugates and their synthesis Additional peptide-lipid conjugates were designed and described in this example, as outlined in Table 3 and Schemes 2-8 below. [Table 3]

[0314] Scheme 2 Synthesis of peptides 9 and 10 [ka] Synthesis of intermediates for peptides 9 and 10 [ka]

[0315] Scheme 2, Step 1: (R)-3-((3-((tert-butoxycarbonyl)amino)propanoyl)oxy)propane-1,2-diyl ditetradecanoate (6). [(2R)-3-Hydroxy-2-tetradecanoyloxy-propyl]tetradecanoate (513 mg, 1 mmol), 3-(tert-butoxycarbonylamino)propanoic acid (227 mg, 1.2 mmol), EDC.HCl (238 mg, 1.3 mmol), and triethylamine (0.21 mL, 1.7 mmol) were mixed in 5 mL of dichloromethane and stirred overnight. It was diluted with another 5 mL of dichloromethane and washed with 1 N HCl (1 × 10 mL) followed by water (1 × 10 mL), dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure. The crude product was purified on a silica gel column (TELEDYNE ISCO Gold, 12 g) using a dichloromethane / ethyl acetate gradient (0-60% over 15 min). The product eluted with a 15-20% ethyl acetate gradient was collected, analyzed and concentrated under reduced pressure to give 540 mg (79%) of pure product. m / z 684.0 (calculated) M-H+Na 706.4 (observed).

[0316] Scheme 2, Step 2: (R)-3-((3-aminopropanoyl)oxy)propane-1,2-diylditetradecanoate (7). The Boc-protected compound [(2R)-3-[3-(tert-butoxycarbonylamino)propanoyloxy]-2-tetradecanoyloxy-propyl]tetradecanoate (500 mg, 0.73 mmol) was taken up in 6 mL of dichloromethane and 4 mL of TFA was added. The mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was purified on a silica gel column using a dichloromethane / methanol gradient (0-60% over 15 min). The product eluted with 20% methanol was collected, concentrated under vacuum and dried to give the pure product (360 mg, 84%), which was used for coupling to the peptide. m / z 583.9 (calculated) M 584.3 (observed).

[0317] Compound 7 can be coupled to the C-terminus of a pre-synthesized STEP peptide sequence that is derivatized at the N-terminus with an acetyl group, and the glutamic acid side chain carboxylic acid is protected with a benzyl ester known from peptide synthesis protocols using Boc-Glu(OBz)-OH using standard coupling agents such as diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) reagents. When Fmoc chemistry is used in peptide synthesis, these amino acid side chains can typically be protected as the tert-butyl ester Fmoc-Glu(OtBu)-OH. Finally, such side chain protecting groups can be removed under hydrogenation conditions or using either formic or trifluoroacetic acid to obtain crude peptides 9 and 10 that can be purified on a C4 column as previously described.

[0318] Scheme 3 Peptides 11 and 12 [ka]

[0319] Synthesis of intermediates for peptides 11 and 12 The intermediate for peptides 11 and 12 is the same as the intermediate for peptides 1-8 provided in Example 1, ie, intermediate 3 shown below. [ka]

[0320] As shown for peptides 11 and 12, peptides can be synthesized containing an additional β-alanine at the C-terminus of each STEP or S(Me)T(Me)QP segment, and the N-terminus of such peptides can be coupled to the three following protocols developed for peptides 1-8 in Example 1 to give crude peptides 11 and 12, which may be purified on a C4 hydrophobic interaction column as described above.

[0321] Scheme 4 Peptides 13 and 14 (cholesterol conjugates) [ka] Following the coupling protocol established for peptides 1-8 in Example 1, commercially available cholesterol NHS hemisuccinate (CAS#88848-79-7) can be used as is in the coupling of the pure peptides to give peptide 13 and peptide 14.

[0322] Scheme 5 Peptides 15 and 16 [ka] Synthesis of intermediates for peptides 15 and 16 [ka] Scheme 5, Step 1: (R)-3-((tert-butoxycarbonyl)amino)propane-1,2-diylditetradecanoate (8). To a solution of tert-butyl N-[(2R)-2,3-dihydroxypropyl]carbamate (0.5 g, 2.6 mmol) in dichloromethane (12 mL) was added tetradecanoic acid (1.8 g, 7.8 mmol), EDC (1.1 g, 5.5 mmol), followed by triethylamine (0.82 mL, 5.9 mmol). The mixture was stirred at room temperature overnight. The solution was diluted with dichloromethane (15 mL) and washed with 1N HCl (2×15 mL), water (2×15 mL), dried (Na 2 SO 4 ), filtered and evaporated under reduced pressure. The residue was purified on a silica gel column using hexane / ethyl acetate. The product was eluted with 30% ethyl acetate. m / z 611.9 (calculated) M-H+Na 634.4 (observed).

[0323] Scheme 5, Step 2: (R)-3-aminopropane-1,2-diylditetradecanoate (9). A solution of IK473 (1.4 g) in 40% TFA (V / V) in dichloromethane was stirred at room temperature for 4 hours. TLC analysis showed the reaction was complete. The solvent was evaporated under reduced pressure and the resulting material was used directly in the next reaction without further purification. m / z 511.8 (calculated) M 512.4 (observed).

[0324] Scheme 5, step 3: (R)-4-((2,3-bis(tetradecanoyloxy)propyl)amino)-4-oxobutanoic acid (10). To a solution of 9 in dichloromethane was added [(2R)-3-amino-2-tetradecanoyloxy-propyl]tetradecanoate followed by tetrahydrofuran-2,5-dione and diisopropylethylamine and the mixture was stirred at room temperature overnight. TLC (10% methanol in dichloromethane) showed two faster moving spots upon iodine / silica gel treatment. Evaporated and loaded onto a TELEDYNE ISCO gold silica gel column and eluted with a 0-60% methanol in dichloromethane gradient over 15 min. Eluted fractions were isolated, analyzed, pooled and evaporated under reduced pressure. m / z 611.9 (calculated) M-H+Na 634.4 (observed).

[0325] Scheme 5, step 4: (R)-3-(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutanamido)propane-1,2-diylditetradecanoate (11). To a solution of 4-[[(2R)-2,3-di(tetradecanoyloxy)propyl]amino]-4-oxo-butanoic acid (404 mg, 0.66 mmol) in 4 mL of dichloromethane was added bis(2,5-dioxopyrrolidin-1-yl)carbonate (338 mg, 1.3 mmol) followed by triethylamine (0.23 mL, 1.7 mmol). The mixture was stirred overnight, diluted with dichloromethane (4 mL), washed with ice-cold water (10 mL), and the dichloromethane solution was isolated and washed with Na 2 SO 4The crude product was dried at 75° C., filtered and evaporated under reduced pressure. The crude product was loaded onto a 12 g Teledyne ISCO gold column with 3 mL of dichloromethane and eluted with a gradient of 0-60% EtOAc in hexanes over 15 min. Product containing fractions were pooled, concentrated under reduced pressure and dried to give 360 ​​mg (77%) of product as a white solid. m / z 709 (calculated) MH 708.1 (observed).

[0326] Intermediate 11 can be used for coupling of peptides at the N-terminus following the protocols developed for peptides 1-8 to give peptide 15 and peptide 16.

[0327] Scheme 6 Peptides 17 and 18 [ka] Synthesis of intermediates for peptides 17 and 18 [ka] Scheme 6, Step 1: (S)-3-(3-(2,3-bis(tetradecanoyloxy)propoxy)-3-oxopropoxy)propanoic acid (12). A mixture of 2 g (3.9 mmol) of [(2R)-3-hydroxy-2-tetradecanoyloxy-propyl]tetradecanoate, 561 mg (2.9 mmol) of EDC.HCl, 0.82 mL (1.5 mmol) of triethylamine, and 474 mg (0.75 mmol) of 3-(2-carboxyethoxy)propanoic acid in 10 mL of dichloromethane was stirred at room temperature overnight. The mixture was diluted with 5 mL of dichloromethane, washed with 10 mL of water followed by brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified on a silica gel column (Teledyne ISCO gold 12 g) with a dichloromethane / ethyl acetate gradient (0-100% ethyl acetate) and the product eluted with 40% ethyl acetate was collected and concentrated under reduced pressure to give 1.2 g (47%) of product. m / z 656.4 (calculated) MH 655.2 (observed).

[0328] Scheme 6, step 2: (S)-3-((3-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropoxy)propanoyl)oxy)propane-1,2-diyl ditetradecanoate (13). A mixture of 3-[3-[(2S)-2,3-di(tetradecanoyloxy)propoxy]-3-oxo-propoxy]propanoic acid (525 mg, 0.80 mmol), bis(2,5-dioxopyrrolidin-1-yl)carbonate (409 mg, 1.6 mmol), and triethylamine (0.28 mL, 2 mmol) in 4 mL of dichloromethane was stirred overnight. The reaction mixture was diluted with dichloromethane (4 mL), washed with ice-cold water (10 mL), and the dichloromethane solution was isolated and cooled to 100° C. for 24 hours. 2 SO 4 The crude product was dried at 75° C., filtered and evaporated. The crude product was loaded onto a 12 g Teledyne ISCO gold column with 3 mL of dichloromethane and eluted with a gradient of 0-60% ethyl acetate in hexanes over 15 min. The product eluted with 20-25% ethyl acetate was collected, concentrated under reduced pressure and dried under vacuum to give 350 mg (58%) of pure product. m / z 754.0 (calculated) M-H+Na 776.2 (observed).

[0329] Intermediate 13 was used in the preparation of peptide 17 and peptide 18, following the coupling and purification protocols used for peptides 1-8 as described in Example 1.

[0330] Peptide 17: HPLC purity 92%. Mass: 2314.7 (calculated), 2314.8 (observed).

[0331] Peptide 18: 100% HPLC purity. Mass: 2422.7 (calculated), 2422.8 (observed).

[0332] Scheme 7 Peptides 19 and 20 [ka] Synthesis of intermediates for peptides 19 and 20 [ka] Scheme 7, Step 1: (R)-4-(2,3-bis(palmitoyloxy)propoxy)-4-oxobutanoic acid (15). To a suspension of 1,2-dipalmitoyl-sn-glycerol (2 g, 3.2 mmol) in 40 mL of anhydrous dichloromethane in a 200 mL RB flask under argon maintained in an ice bath, 563 mg (5.6 mmol) of succinic anhydride was added, followed by 902 mg (7.4 mmol) of DMAP. The mixture was allowed to reach room temperature and stirred overnight at room temperature. TLC analysis (10% methanol / dichloromethane) showed a slower moving spot with DMAP at the bottom. The mixture was washed with 1 N HCl (3 x 30 mL), water, and brine (100 mL each), dried (Na 2 SO 4 ), filtered and evaporated. Column purification (Teledyne ISCO 40g) using a methanol / dichloromethane gradient elution product at 12-15% methanol. Concentration of the fractions gave 2g (85%) of product as a white solid. m / z 668.5 (calculated) MH 667.5 (observed).

[0333] Scheme 7, step 2: (R)-2,3-Bis(palmitoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate (16). To a mixture of 4-[(2R)-2,3-di(hexadecanoyloxy)propoxy]-4-oxo-butanoic acid (2 g, 3 mmol), triethylamine (0.83 mL, 6 mmol) and DMAP (50 mg, cat.) in 40 mL anhydrous dichloromethane, bis(2,5-dioxopyrrolidin-1-yl)carbonate (1.15 g, 4.5 mmol) was added and the mixture was stirred at room temperature overnight. The reaction was quenched by adding 2 equivalents of acetic acid. The mixture was diluted with dichloromethane and washed with ice-cold water (2×80 mL) followed by brine (80 mL), dried (Na 2 SO 4) and evaporated under reduced pressure. The residue was purified on a silica gel column (Teledyne ISCO 40) using a dichloromethane:ethyl acetate gradient (0-40% over 30 min). The product was eluted with 10-12% ethyl acetate. The solvent was removed on a rotary evaporator and the resulting white solid was dried under vacuum to give 1.6 g of product. m / z 765.5 (calculated) M+H 788.5 (observed).

[0334] Intermediate 16 was used in the preparation of peptide 19 and peptide 20, following the coupling and purification protocols used for peptides 1-8 as described in Example 1.

[0335] Peptide 19: Mass: 2326.8 (calculated), 2326.0 (observed).

[0336] Peptide 20: Mass: 2434.8 (calculated), 2434.0 (observed).

[0337] Scheme 8 Peptides 21 and 22 [ka] Synthesis of intermediates for peptides 21 and 22 [ka] Scheme 8, Step 1: (R)-4-(2,3-bis(stearoyloxy)propoxy)-4-oxobutanoic acid (18). To a suspension of (S)-3-hydroxypropane-1,2-diyldistearic acid (2 g, 3.2 mmol) in 40 mL of anhydrous dichloromethane in a 200 mL RB flask under argon maintained in an ice bath, 512 mg (5.6 mmol) of succinic anhydride was added, followed by 821 mg (7.4 mmol) of DMAP. The mixture was allowed to reach room temperature and stirred overnight at room temperature. TLC analysis (10% methanol / dichloromethane) showed a slower moving spot with DMAP at the bottom. The mixture was washed with 1 N HCl (3×30 mL), water, and brine (100 mL ethyl acetate), dried (Na 2 SO 4 ), filtered and evaporated. Column purification (Teledyne ISCO 80g) using a methanol / dichloromethane gradient elution product at 12-15% methanol. Concentration of the fractions gave 2g (86%) of product as a white solid. m / z 724.5 (calculated) MH 723.5 (observed).

[0338] Step 2: (R)-2,3-Bis(stearoyloxy)propyl(2,5-dioxopyrrolidin-1-yl)succinate (19). To a mixture of (R)-4-(2,3-bis(stearoyloxy)propoxy)-4-oxobutanoic acid (2 g, 2.8 mmol), triethylamine (0.77 mL, 5.5 mmol) and DMAP (50 mg, cat.) in 30 mL anhydrous dichloromethane, bis(2,5-dioxopyrrolidin-1-yl)carbonate (1.1 g, 4.1 mmol) was added and the mixture was stirred at room temperature overnight. The reaction was quenched by the addition of 2 equivalents of acetic acid. The mixture was diluted with dichloromethane and washed with ice-cold water (2×80 mL) followed by brine (80 mL), dried (Na 2 SO 4) and evaporated under reduced pressure. The residue was purified on a silica gel column (Teledyne ISCO 40) using a dichloromethane:ethyl acetate gradient (0-40% over 30 min). The product was eluted with 10-12% ethyl acetate. The solvent was removed under rotary evaporation and the resulting white solid was dried to give 1.5 g (66%) of product. m / z 822.5 (calculated) M+Na 845.5 (observed).

[0339] Intermediate 19 was used to prepare peptide 21 and peptide 22, following the coupling and purification protocols used for peptides 1-8 as described in Example 1.

[0340] Peptide 21: Mass: 2382.9 (calculated), 2382.0 (observed).

[0341] Peptide 22: Mass: 2490.9 (calculated), 2491.0 (observed).

[0342] Abbreviations used DCM: dichloromethane DMAP: N,N-dimethylpyridine DMG: Dimyristoyl glycerol DPG: Dipalmitoyl glycerol DSG: distearoyl glycerol EA: Ethyl acetate EDC.HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HCl: Hydrochloric acid TEA: Triethylamine TFA: Trifluoroacetic acid TLC: Thin Layer Chromatography

[0343] Example 10: Further peptide-lipid conjugates and their synthesis Selected peptide-lipid conjugates from Example 9 were formulated into lipid nanoparticles and characterized according to the methods and protocols described in Example 2. The lipid nanoparticles showed good particle size, dispersion, and encapsulation, as shown in the data in Table 4 below. These lipid nanoparticle formulations included an ionic cationic lipid ("Cat"), a helper lipid (distearoylphosphatidylcholine, "DSPC"), cholesterol ("Chol"), and the lipid-peptide conjugates shown. The ionic cationic lipids used in these formulations were selected to provide a common lipid that can serve as a basis for comparison, but one of skill in the art will recognize that the lipid-peptide conjugates of the present disclosure can be combined with any cationic lipid suitable for use in lipid nanoparticle formulations for delivery of active agents, such as nucleic acids. The ionic cationic lipids used in these formulations have the following structure: [ka] has.

[0344] Lipid nanoparticle formulations were prepared and characterized as described in Example 4, and details of each formulation, along with the resulting properties, are provided in Table 2 below. In this table, "N / P" refers to the ratio of cationic amino groups from the ionizable cationic lipid to the anionic phosphate backbone groups of the encapsulated nucleic acid. The results show that the peptide-lipid conjugates of the present disclosure are successfully integrated into lipid nanoparticle formulations with good particle size, polydispersity, and percent encapsulation of nucleic acid.

[0345] The formulations are further tested for in vivo measurement of hEPO expression according to the protocol outlined in Example 7. [Table 4]

[0346] Further considerations The above examples demonstrate that peptide-lipid conjugates are at least suitable, and in some examples, a superior alternative to PEG-lipids in lipid-formulated nucleic acid delivery systems. These peptide-lipid conjugates have shown comparable or superior levels of delivery of mRNA for in vivo expression and siRNA for target knockdown. When peptide-lipid conjugates further comprise a threshold amount of hydrophilic amino acids as described above, they provide the additional benefit of being able to formulate lipid compositions in aqueous media of suitable morphology, size, and dispersion. These hydrophilic side chains allow the peptide portion of the lipid conjugate at the outer surface of the composition to maximize the association of the peptide with the aqueous media, thereby providing a more ideal conformation (e.g., "water cage"). In addition, when peptide-lipid conjugates have a threshold amount of proline, they provide additional conformational benefits by allowing the right amount of structural rigidity for the peptide portion of the conjugate. Similarly, minimizing glycine can provide the advantage that the peptide portion of the peptide-lipid conjugate will have less variability in overall conformation and will only generate conformations that are more favorable for maintaining uniform morphology, size, and distribution of the resulting composition.

[0347] The foregoing description is provided to enable one skilled in the art to practice the various configurations described herein. While the subject technology has been specifically described with reference to various figures and configurations, it should be understood that these are for illustrative purposes only and should not be construed as limiting the scope of the subject technology.

[0348] Furthermore, to the extent that the terms "include," "have," or similar terms are used in the description or claims, such terms are intended to be inclusive in the same manner as the term "comprise" when it is used as a transitional term in a claim.

[0349] In one or more embodiments, the terms "about," "substantially," and "approximately" may provide for an industry-accepted tolerance, such as less than 1 percent to 5 percent, for their corresponding terms and / or relativities between items.

[0350] Reference to a singular element is not intended to mean "one and only one" unless otherwise specified, but rather "one or more". Masculine pronouns (e.g., his) include feminine and neuter genders (e.g., hers and its), and vice versa. The term "some" refers to one or more. Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject art, and are not referred to in connection with interpreting the description of the subject art. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure that are known or later become known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the subject art. Furthermore, any disclosures disclosed herein are not intended to be directed to the general public, regardless of whether such disclosures are expressly recited in the description above.

[0351] Although the detailed description includes many details, these should not be construed as limiting the scope of the subject technology, but merely as illustrating different examples and aspects of the subject technology. Naturally, the scope of the subject technology includes other embodiments not discussed in detail above. Various other modifications, changes, and variations may be made in the arrangement, operation, and details of the methods and apparatus of the subject technology disclosed herein without departing from the scope of the present disclosure. Unless otherwise stated, reference to an element in the singular is not intended to mean "only one" unless expressly stated, but rather to mean "one or more". In addition, a composition or method need not address every problem that can be solved (or have every advantage that can be achieved) by different embodiments of the present disclosure to be encompassed within the scope of the present disclosure. The use of "can" and its derivatives herein should be understood in the sense of "possibly" or "optionally", as opposed to positive ability.

Claims

1. A lipid composition containing a nucleic acid, comprising a peptide-lipid conjugate, the peptide of the peptide-lipid conjugate consists of 8 to 52 amino acids, and at least 25% of the amino acids in the peptide of the peptide-lipid conjugate are proline; The peptide-lipid conjugate comprises 0.1 mol% to 4 mol% of all lipids in the lipid composition; Lipid composition.

2. The lipid composition described in claim 1, wherein 25% to 60% of the amino acids in the peptide of the peptide-lipid conjugate are proline.

3. The lipid composition of claim 1, wherein 28% to 80% of the amino acids in the peptide of the peptide-lipid conjugate have hydrophilic side chains.

4. The amino acid having a hydrophilic side chain is selected from the group consisting of glutamine, glutamic acid, asparagine, aspartic acid, serine, O—C １－６ Alkylserine, threonine, and O—C １－６ 4. The lipid composition of claim 3, wherein the lipid composition is any combination of amino acids selected from alkylthreonine.

5. The lipid composition of claim 1, wherein less than 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine.

6. The lipid composition of claim 1 , wherein the peptide does not contain glycine.

7. 2. The lipid composition of claim 1, wherein the lipid composition is selected from lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions.

8. The lipid composition of claim 1 , wherein the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol.

9. The lipid composition of claim 1, wherein the peptide-lipid conjugate comprises 1 mol% of all lipids in the lipid composition.

10. The lipid composition of claim 1 , wherein the lipid composition encapsulates the nucleic acid.

11. The lipid composition of claim 1, wherein the lipid of the peptide-lipid conjugate is conjugated to the peptide via a linker.

12. The linker may be an amide (—C(O)NH—), an amino (—NR Ｎ -), wherein R Ｎ But H, C １－６ Alkyl, carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (-(O)CCH ２ CH ２ C(O)-), succinamidyl (-NHC(O)CH ２ CH ２ 12. The lipid composition of claim 11, wherein the lipid is selected from the group consisting of ethers, carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), and sulfonate esters.

13. The lipid of the peptide-lipid conjugate is didecyloxypropyl (C １０ ), dilauryloxypropyl (C １２ ), dimyristyloxypropyl (C １４ ), dipalmityloxypropyl (C １６ ), or distearyloxypropyl (C １８ ), 1,2-dipalmityloxypropyl-3-amine (DOMG), 1,2-dimyristyloxypropylamine (DMG), 1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine (DLPE), dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), and cholesterol or a cholesterol derivative.

14. The peptide is conjugated at its C-terminus to the lipid of the peptide-lipid conjugate, and the amino group at the N-terminus of the peptide is substituted with one or two C １－６ The lipid composition of claim 1 , substituted with an alkyl group or an amide group.

15. The peptide is conjugated at its N-terminus to the lipid of the peptide-lipid conjugate, and the amino acid at the C-terminus of the peptide is alkylated to form a C １－６ The lipid composition of claim 1 , which forms an alkyl ester or is amidated.

16. 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammoniumpropane-(DODAP), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1 , 2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-Dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-Distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-Dimyrityloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-Dimethylamino-2-(cholest-5-ene-3-beta-oxybutane) -4-oxy)-1-(cis,cis-9,12-oc-tadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropyl 2. The lipid composition of claim 1, further comprising one or more cationic lipids selected from DLinDAP, 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or (DLin-K-XTC2-DMA).

17. The lipid composition of claim 1 , further comprising one or more helper lipids.

18. A lipid composition comprising a peptide-lipid conjugate and a nucleic acid, a. the peptide of the peptide-lipid conjugate consists of 8 to 52 amino acids; b. at least 14% of the amino acids in the peptide of the peptide-lipid conjugate are proline; c. 28% to 80% of the amino acids in the peptide of the peptide-lipid conjugate have hydrophilic side chains; d. less than 43% of the amino acids in the peptide of the peptide-lipid conjugate are glycine; e. the lipid composition comprises one or more cationic lipids, one or more helper lipids, and a sterol; f. A lipid composition, wherein the peptide-lipid conjugate comprises 0.1 mol % to 10 mol % of all lipids in the lipid composition.

19. The lipid composition of claim 1 , wherein the nucleic acid is selected from siRNA, antisense oligonucleotides, UNA oligomers, mRNA, microRNA, and DNA.

20. 20. The lipid composition of claim 19, wherein the mRNA is a self-replicating mRNA.

21. A medicament for treating a disease in a subject in need thereof, comprising the lipid composition of claim 1.

22. An agent for expressing a protein or polypeptide of interest in a cell, comprising the lipid composition of claim 1, wherein the nucleic acid is mRNA or self-replicating mRNA.

23. A vaccine comprising the lipid composition of claim 1, wherein the nucleic acid is mRNA or a self-replicating mRNA.

24. An agent for inducing an immune response in a subject, comprising the vaccine of claim 23.

25. The lipid composition described in claim 3, wherein the amino acids having hydrophilic side chains are a combination of glutamic acid, serine, and threonine.

26. The lipid composition described in claim 3, wherein the amino acids having hydrophilic side chains are a combination of methylserine, methylthreonine, and glutamine.

27. The lipid composition of claim 1 or 18, wherein the peptide of the peptide-lipid comprises at least one unit of a STEP tetrapeptide or an S(Me)T(Me)QP tetrapeptide.

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