WO2023075527A1 - Complex of lipid nanoparticles and novel cell-penetrating peptides - Google Patents

Abstract

The present invention relates to a complex of a lipid nanoparticle and a novel cell-penetrating peptide, and, more specifically, to a complex comprising a lipid nanoparticle and a novel peptide having better cell-penetrating property than common TAT peptides. The complex can effectively penetrate into cells, tissues, blood and the like of a living body.

Description

Complex of lipid nanoparticles and cell-permeable novel peptides

This application claims priority to Republic of Korea Patent Application No. 10-2021-0146624 filed on October 29, 2021, and the entire specification is a reference in this application.

It relates to a complex of a lipid nanoparticle and a new peptide having cell permeability, and more particularly, to a complex including a lipid nanoparticle containing a nucleic acid and a novel peptide exhibiting improved cell permeability compared to a commercially available TAT peptide.

Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds such as cell impermeable therapeutic nucleic acids, proteins, and peptides. For example, nucleic acid-based drugs that include large nucleic acid molecules, such as in vitro transcribed messenger RNA (mRNA), as well as smaller polynucleotides that interact with messenger RNA or genes, must be delivered to the appropriate cellular compartment to be effective. . Double-stranded nucleic acids such as double-stranded RNA molecules (dsRNA), including siRNA, suffer from cell penetration due to their physicochemical properties. When delivered to the appropriate compartment, siRNA blocks gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). In general, siRNAs are large, with molecular weights ranging from 12 to 17 kDa, and are highly anionic due to their phosphate backbones with up to 50 negative charges. Also, two complementary RNA strands create a rigid helix. Due to these features, siRNAs have poor drug likeness. In addition, siRNAs are rapidly degraded by nucleases present in blood and other body fluids or tissues and have been shown to stimulate strong immune responses in vitro and in vivo (Robbins et al., Oligonucleotides 19:89-102, 2009). mRNA molecules suffer similar problems of impermeability, fragility and immunogenicity.

However, lipid nanoparticle formulations have improved nucleic acid delivery in vivo. Such formulations, for example, significantly reduced the siRNA dose required to achieve target knockdown in vivo (Zimmermann et al., Nature 441:111-114, 2006). Typically, such lipid nanoparticle drug delivery systems are multi-component formulations comprising a cationic lipid, a helper lipid, and a lipid comprising polyethylene glycol. Positively charged cationic lipids bind to anionic nucleic acids, while other components support stable self-assembly of lipid nanoparticles.

Efforts have been made to improve the delivery efficacy of such lipid nanoparticle formulations. Many such efforts are aimed at developing more suitable cationic lipids. Despite these efforts, there remains a need for formulations comprising lipid nanoparticles that provide high post-administration potency and enable administration of lower doses of nucleic acids.

On the other hand, in general, biologically active substances made of polymers such as proteins and DNA cannot pass through a hydrophilic and hydrophobic phospholipid bilayer and cannot pass through cell membranes and enter cells. However, cell penetrating peptides are known that can cross cell membranes without the help of other molecules such as receptors.

Cell penetrating peptides, also called CPP (Cell penetrating peptide) and MTS (membrane translocating sequences), pass through the cell membrane in a form bound to a transport target or in a mixed form to transport targets such as proteins, DNA, RNA, and lipid particles into cells. It can also be transported into the cytoplasm, organelles, and nucleus.

As one of the infection mechanisms of HIV-1 (Human immunodeficiency virus-1), it is the first protein to be confirmed that a substance called TAT penetrates the cell membrane.

In addition, Penetratin (Antp), a cell-penetrating peptide composed of 16 amino acid sequences derived from Antennapedia homeoprotein, an essential transcription factor for the development of Drosophila, derived from VP22, a protein expressed by HSV-1 (Herpes simplex virus type 1) Cell-penetrating peptides such as VP22, the cell-penetrating peptide of the same name, Transportan consisting of artificially synthesized 27 amino acid sequences, and Poly-Arginine, an artificially repeated arginine expected to play the most important role in cell-penetrating peptides. is well known as

These conventional cell-penetrating peptides are sequences derived from viral proteins such as HIV-1, or derived from proteins expressed by other species such as Drosophila, or amino acid sequences constituting conventional cell-penetrating peptides are analyzed to determine a characteristic amino acid sequence. Since it is an amino acid sequence that was selected and artificially synthesized, it could cause side effects such as an immune response when applied to the human body.

In addition, since they consist of relatively long amino acid chains, they are more likely to cause unwanted immune responses and can affect the structure and function of the protein to be delivered, so they are less efficient when linked with biologically active substances to be delivered into cells. There was a problem with deterioration.

On the other hand, recently, experiments for introducing therapeutic drugs, peptides, and proteins into cells have been actively conducted. However, all of these methods have a limitation in that they can deliver nucleic acids only to some cells, and there is a problem of toxicity by damaging cell membranes.

Therefore, as a result of intensive research to develop a method for effectively delivering lipid nanoparticles containing nucleic acids into cells, the present inventors have developed CPP variants derived from TCTP protein (translationally controlled tumor protein) representing a series of sequences into lipid nanoparticles. As a result of binding to the particles, it was confirmed that the stability of the nucleic acid in the cytoplasm was greatly improved, and the present invention was completed.

Accordingly, an object of the present invention is to provide a complex comprising a lipid nanoparticle and a cell penetrating peptide consisting of the following amino acid sequence:

MIIFR-R1-R2-R3-R4-R5-R6

In the above formula,

R1 is any one amino acid selected from the group consisting of A, V, I, L, S, F, K and R,

R2 is any one amino acid selected from the group consisting of S, L, F, T and Y;

R3 is any one amino acid selected from the group consisting of E, L, A and R,

R4 is any one amino acid selected from the group consisting of Q, H, T, L, D and R,

R5 is any one amino acid selected from the group consisting of L, S, V, A, K and H;

R6 is any one dipeptide selected from the group consisting of DK, EK, QK, NK, KK and FK.

Another object of the present invention is to provide a composition for promoting cellular permeation of a substance containing a lipid nanoparticle and a peptide complex.

Another object of the present invention is to provide a composition for accelerating cell permeation of a substance composed of a lipid nanoparticle and a peptide complex.

Another object of the present invention is to provide a composition for accelerating cell permeation of a substance essentially consisting of a lipid nanoparticle and a peptide complex.

Another object of the present invention is to provide a use of a material comprising the lipid nanoparticle and the peptide complex for preparing a composition for promoting cell permeation.

Another object of the present invention is to provide a method for promoting cell permeation comprising administering an effective amount of a composition containing the lipid nanoparticle and a peptide complex-containing material as an active ingredient to a subject in need thereof.

In order to achieve the above object, the present invention provides a complex comprising a lipid nanoparticle and a cell penetrating peptide consisting of the following amino acid sequence:

MIIFR-R1-R2-R3-R4-R5-R6

In the above formula,

R1 is any one amino acid selected from the group consisting of A, V, I, L, S, F, K and R,

R2 is any one amino acid selected from the group consisting of S, L, F, T and Y;

R3 is any one amino acid selected from the group consisting of E, L, A and R,

R4 is any one amino acid selected from the group consisting of Q, H, T, L, D and R,

R5 is any one amino acid selected from the group consisting of L, S, V, A, K and H;

R6 is any one dipeptide selected from the group consisting of DK, EK, QK, NK, KK and FK.

In order to achieve another object of the present invention, the present invention provides a composition for promoting cell permeation of a substance containing a lipid nanoparticle and a peptide complex.

In addition, the present invention provides a composition for accelerating cell permeation of a substance composed of lipid nanoparticles and peptide complexes.

In addition, the present invention provides a composition for accelerating cell permeation of substances essentially consisting of lipid nanoparticles and peptide complexes.

In order to achieve another object of the present invention, the present invention provides the use of a material containing the lipid nanoparticle and the peptide complex for preparing a composition for promoting cell permeation.

In order to achieve another object of the present invention, the present invention provides a method for promoting cell permeation comprising administering an effective amount of a composition containing as an active ingredient a substance containing the lipid nanoparticle and the peptide complex to a subject in need thereof to provide.

Hereinafter, the present invention will be described in detail.

The practice of the present invention, unless specifically indicated to the contrary, uses conventional methods of molecular biology and recombinant DNA technology within the technical field to which the present invention pertains, and for illustrative purposes is largely known in the art.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Throughout the disclosure herein, various aspects or conditions relating to the present invention may be suggested in a range format. In this specification, the description of a range value is meant to include a corresponding boundary value, that is, to include all values from the lower limit value to the upper limit value unless otherwise specified. It is to be understood that the description of range formats is merely for convenience and brevity and is not to be construed as an inflexible limitation on the scope of the invention. Accordingly, statements of ranges should be considered to have specifically disclosed all possible subranges as well as individual numerical values within the range. For example, the description of a range, such as 7 to 170, can include individual values within the range, such as 10 to 127, 23 to 35, 80 to 100, 50, as well as 9, 27, 35, 101, and 155. to 169, etc., should be considered as specifically disclosed. This applies regardless of the width of the range.

The term 'comprising' of the present invention is used the same as 'containing' or 'characterized by', and does not exclude additional components or method steps not mentioned in the composition or method. .

As used herein, the terms 'peptide' and 'protein' are used according to their usual (conventional) meaning, that is, they refer to an amino acid sequence. A peptide is not limited to a particular length, but in the context of the present invention generally refers to a fragment of a full-length protein and is subject to post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., and others known in the art. It may contain modifications (both naturally occurring and non-naturally occurring modifications) and may be referred to as a "polypeptide". The peptides and proteins of the present invention can be prepared using any of a variety of known recombinant and/or synthetic techniques, illustrative examples of which are further described below.

The present invention provides a complex comprising a lipid nanoparticle and a cell penetrating peptide consisting of the following amino acid sequence:

MIIFR-R1-R2-R3-R4-R5-R6

In the above formula,

R1 is any one amino acid selected from the group consisting of A, V, I, L, S, F, K and R,

R2 is any one amino acid selected from the group consisting of S, L, F, T and Y;

R3 is any one amino acid selected from the group consisting of E, L, A and R,

R4 is any one amino acid selected from the group consisting of Q, H, T, L, D and R,

R5 is any one amino acid selected from the group consisting of L, S, V, A, K and H;

R6 is any one dipeptide selected from the group consisting of DK, EK, QK, NK, KK and FK.

The peptide of the present invention is a novel peptide having cell membrane permeability, which itself has excellent cell membrane permeability.

In the present invention, the "cell membrane" means a membrane that builds a boundary between a cell and the outside of the cell. As one aspect of the present invention, the cell membrane may be a cell membrane of any organism, for example, a unicellular or multicellular organism belonging to bacteria (Bacteria), archaea (Archaea) and eukaryote (Eukarya). The eukaryote may be any organism belonging to the protista, fungus, plant, and animal kingdoms. The animal may be any animal including humans.

In the present invention, the "cell membrane" may be a membrane of any type of cell. For example, the "cell" may be selected from the group consisting of epithelial cells, muscle cells, immune cells and endothelial cells, but is not limited thereto. The cells form the lining of an organ directly in contact with the outside of the body, such as endothelium, epithelium, or mucous membrane, or are cells of a cell layer covering the surface of an organ or blood vessel in the body. It can be. In one aspect of the present invention, the cells include skin epithelial cells, hair follicle epithelial cells, mucosal epithelial cells, corneal epithelial cells, scalp epithelial cells, corneal endothelial cells, and vascular endothelial cells. For example, the mucosal epithelial cells may be at least one mucosal epithelial cell selected from the group consisting of nasal cavity, lung, vagina, rectum, anus, urethra, sublingual, ocular, conjunctival and oral mucosa. For example, the vascular endothelial cells may be endothelial cells of various arteries, veins, and capillaries, including cerebral vessels constituting the blood-brain barrier (BBB), endothelial cells of lymphatic vessels, and endothelial cells of the lining of the heart. In addition, the cells may be various cancer cells including cervical cancer, breast cancer, liver cancer, lung cancer, and the like. In addition, the cells may be cells having receptors for various substances such as hormones, chemical transmitters in the nervous system, and drugs. For example, insulin receptors, glucagon-like-protein 1 (Glucagon-like- It may be a cell having a protein 1, GLP1) receptor, but the cell is not limited to the above examples.

The peptides of the present invention can be prepared using available techniques known in the art. For example, it can be prepared using any of a variety of proteolytic enzymes. Exemplary proteases (proteinases) include, for example, achromopeptidase, aminopeptidase, ancrod, angiotensin converting enzyme, bromelain , calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B, carboxypeptidase G G), carboxypeptidase P, carboxypeptidase W, carboxypeptidase Y, caspase 1, caspase 2, caspase caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9 (caspase 9), caspase 10 (caspase 10), caspase 11 (caspase 11), caspase 12 (caspase 12), caspase 13 (caspase 13), cathepsin B (cathepsin B), cathepsin C C), cathepsin D, cathepsin E, cathepsin G, cathepsin H, cathepsin L, chymopapain, key chymase, chymotrypsin, clostripain, collagenase, complement C1r, complement C1s, complement Factor D, complement Complement factor I, cucumisin, dipeptidyl peptidase IV, elastase (leukocyte), pancreatic elastase (pancreatic), endoproteinase Arg-C (endoproteinase Arg-C), endoproteinase Asp-N (endoproteinase Asp-N), endoproteinase Glu-C (endoproteinase Glu-C), endoproteinase Lys-C ( endoproteinase Lys-C), enterokinase, factor Xa, ficin, furin, granzyme A, granzyme B, HIV protease ), IGase, kallikrein tissue, leucine aminopeptidase, general, cytosol leucine aminopeptidase, microsomal leucine aminopeptidase, microsomal), matrix metalloprotease, methionine aminopeptidase, neutrase, papain, pepsin, plasmin, prolidase , pronase E, prostate specific antigen, protease alkalophilic from Streptomyces griseus, protease from Aspergillus, protease from Aspergillus saitoi ( protease from Aspergillus saitoi), protease from Aspergillus sojae, B. licheniformis protease (protease B. licheniformis, alkaline or alcalase), protease from Bacillus polymyxa, protease from Bacillus sp (protease from Bacillus sp), protease from Rhizopus sp., protease S (protease S), proteasomes, proteinase from Aspergillus oryzae (proteinase from Aspergillus oryzae), Proteinase 3, proteinase A, proteinase K, protein C, pyroglutamate aminopeptidase, renin ( rennin), streptokinase, subtilisin, thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptase and urokinase. A person skilled in the art can easily determine which proteolytic enzyme is appropriate, taking into account the chemical specificity of the fragment to be produced.

The peptides of the present invention can be prepared by any suitable procedure known to those skilled in the art, such as recombinant techniques. In addition to recombinant production methods, the polypeptides of the present invention can be prepared by direct peptide synthesis using solid phase techniques.

In the solid-phase peptide synthesis (SPPS) method, synthesis can be initiated by attaching functional units, called linkers, to small porous beads to guide the peptide chain. Unlike the liquid-phase method, the peptide is covalently bound to the beads and prevents them from being separated by filtration until they are cleaved by a specific reactant, such as trifluoroacetic acid (TFA). A protection process in which the N-terminal amine of a peptide attached to a solid phase is combined with an N-protected amino acid unit, a deprotection process, a re-exposed amine group and a new Synthesis is performed by repeating a cycle (deprotection-wash-coupling-wash) of a coupling process in which amino acids are combined. The SPPS method can be performed using microwave technology together, and microwave technology can shorten the time required for coupling and deprotection of each cycle by applying heat in the peptide synthesis process. The thermal energy may prevent folding or aggregation of the extended peptide chain and promote chemical bonding.

In addition, the peptide of the present invention can be prepared by a liquid phase peptide synthesis method, and the specific method thereof is referred to the following documents: US Patent No. 5,516,891. In addition, the peptide of the present invention can be synthesized by various methods such as a method of mixing the solid phase synthesis method and the liquid phase synthesis method, and the preparation method is not limited to the means described herein.

Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished using, for example, an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments can be chemically synthesized separately and combined using chemical methods to produce the target molecule.

In the present invention, the peptide is a D-form or L-form, a peptide composed of only a part of the D-form or L-form sequence, or all of them in the form of a racemate through a conventional peptide synthesis method or preparation method known to those skilled in the art. can be produced and used. In addition, other conventional modifications known in the art are possible to increase the stability of the peptide.

In the present invention, the peptide may include conventional variants. The variant is one in which an arbitrary change has occurred in the amino acid sequence of the 'peptide', and may include one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring, or may be any of a number of techniques well known in the art, for example, by modifying or altering one or more of the above peptide sequences of the present invention and producing those described herein. It can be produced synthetically by evaluating biological activity.

In one aspect of the invention, the variant comprises conservative substitutions. A 'conservative substitution' is a substitution in which an amino acid is substituted with another amino acid having similar properties, and a person skilled in the art can predict that the secondary structure and hydropathic nature (hydropathic nature, hydrophobic or hydrophilic nature) of the peptide are substantially unchanged. . In general, the following groups of amino acids exhibit conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Modifications may be performed within the structure of the peptides of the present invention, yielding functional molecules that encode peptide variants or derivatives having desired (desirable) characteristics. If it is desired to change the amino acid sequence of the peptide in order to prepare an equivalent or improved variant of the peptide of the present invention, one or more codons can be changed based on protein codon information known in the art by those skilled in the art. there is.

In addition, the variant may contain non-conservative alterations. In a preferred embodiment, variant peptides may differ from the native sequence by substitutions, deletions or additions of 5 or fewer amino acids. Variants can also be altered, for example by deletion or addition of amino acids that have minimal impact on the secondary structure and hydropathic properties of the peptide.

In the present invention, the peptide may include a signal (or leader) sequence at the N-terminus of the protein, and this sequence directs the transfer of the protein simultaneously or after translation. The peptide may also be conjugated with a linker sequence or other sequence to facilitate synthesis, purification or identification of the peptide (eg poly His), or to enhance binding of the peptide to a solid support. there is.

In the present invention, 'complex' means that one or more lipid nanoparticles are included in one or more peptides. In the present invention, the peptide and lipid nanoparticles in the "complex" may be included in a ratio of 1: 1, 1: Da, Da: 1, Da: Da. For example, the peptide and the lipid nanoparticle may be included in a molar ratio of 1:1 to 1:100, or 100:1 to 1:1. However, the molar ratio is exemplary and is not limited thereto. In the present invention, the complex may be formed by simply mixing the peptide and the lipid nanoparticles, by mixing the peptide and lipid nanoparticles, or by linking them by a chemical bond, in particular, the peptide and lipid Nanoparticles may be conjugated.

In one aspect of the present invention, the peptide is conjugated to form a complex with a lipid nanoparticle comprising:

(i) one or more nucleic acid molecules; (ii) cholesterol; (iii) DSPC; (iv) PEG-C-DNA; and (v) a cationic lipid of Formula 1:

[Formula 1]

or as a salt thereof,

where the molar percentages of total lipids relative to PEG-C-DNA, cationic lipids, cholesterol, and DSPC are:

PEG-C-DNA: about 1.5;

Cationic lipids: about 50.0;

Cholesterol: about 38.5; and

DSPC: around 10.0.

In the present invention, the lipid refers to a group of organic compounds, including but not limited to esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. They generally fall into at least three classes: (1) 'simple lipids', which include waxes as well as fats and oils; (2) 'complex lipids' comprising phospholipids and glycolipids; and (3) 'derived lipids' such as steroids.

Lipid particles (eg, LNPs) in the present invention are typically 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, or about 70 nm. to about 90 nm in average diameter, and is substantially non-toxic. In addition, the nucleic acids, when present in the lipid particles of the invention, are resistant in aqueous solution to degradation due to nucleases. Nucleic acid-lipid particles and methods of making them are disclosed, for example, in US Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

In the present invention, the lipid nanoparticle (LNP) refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle (eg, a stable nucleic acid-lipid particle). LNP refers to particles made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids that prevent particle aggregation), nucleic acids, wherein the nucleic acids (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self-amplifying RNA, or plasmid, including interfering RNA or plasmids from which mRNA is transcribed) are encapsulated in lipids. Nucleic acids are at least 50%, 75%, 90%, 100% encapsulated in lipids. LNPs typically include cationic lipids, non-cationic lipids, and lipid conjugates (eg, PEG-lipid conjugates). LNPs can have a prolonged circulatory life following intravenous (i.v.) injection and can accumulate at distal sites (eg, at sites physically separate from the site of administration) where expression or targeting of transfected genes occurs. It is very useful for systemic application as it can mediate the silencing of gene expression.

In the present invention, the nucleic acid refers to a polymer comprising at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA and RNA. DNA includes, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups and It may be in the form of a combination. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids that are synthetic, naturally occurring, and non-naturally occurring, and that contain known nucleotide analogs or modified framework residues or linkages that have similar binding properties to reference nucleic acids. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). do. Unless specifically limited, the term includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties to standard nucleic acids. Unless otherwise indicated, a particular nucleic acid sequence also implies conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences thereof, as well as explicitly indicated sequences. to include Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and/or deoxyinosine residues. A "nucleotide" includes the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other through phosphate groups. “Base” refers to the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and purines and pyrimidines, further including their natural analogues, and including but not limited to amines, alcohols, thiols, carboxylates, and alkylhalides. synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups such as

The term "interfering RNA" or the term "RNAi" or "interfering RNA sequence" refers to when the interfering RNA is in the same cell as the target gene or sequence (e.g., mediates degradation of mRNA complementary to the interfering RNA sequence or inhibits translation). single-stranded RNA (eg, mature miRNA) or double-stranded RNA (eg, duplex RNA such as siRNA, aiRNA, or pre-miRNA) capable of reducing or inhibiting the expression of a target gene or sequence by . Interfering RNA thus refers to a single-stranded RNA that is complementary to a target mRNA sequence or to a double-stranded RNA formed by two complementary strands or by a single self-complementary strand. An interfering RNA may have substantial or complete identity to a target gene or sequence, or may have regions of mismatch (ie, mismatch motifs). The sequence of the interfering RNA may correspond to a full-length target gene or a subsequence thereof.

An interfering RNA is a "small-interfering RNA" or "siRNA", e.g., about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and is double stranded. siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length) . The siRNA duplex may include a 3' overhang and a 5' phosphate end of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides. Examples of siRNAs include, without limitation, double-stranded polynucleotide molecules assembled from two separate stranded molecules, wherein one strand is the sense strand and the other strand is the complementary antisense strand; A double-stranded polynucleotide molecule assembled from single-stranded molecules, wherein sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; It comprises a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; A circular single-stranded polynucleotide molecule with a stem having two or more loop structures and self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

The siRNA molecules described herein may have a 3' overhang of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may have no overhang on one or both sides of the double-stranded region. (i.e., with blunt ends). Preferably, the siRNA has a 3' overhang of two nucleotides on each side of the double-stranded region. In certain instances, the 3' overhang of the antisense strand is complementary to the target sequence and the 3' overhang of the sense strand is complementary to the complementary strand of the target sequence. Alternatively, the 3' overhang has no complementarity to the target sequence or its complementary strand. In some embodiments, the 3' overhang comprises one, two, three, four, or more nucleotides, such as a 2'-deoxy (2'H) nucleotide. In certain preferred embodiments, the 3' overhang comprises deoxythymidine (dT) and/or uridine nucleotides. In another embodiment, one or more of the nucleotides in the 3' overhang on one or both sides of the double stranded region comprises a modified nucleotide. Non-limiting examples of modified nucleotides are described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxy nucleotides, 2'-O-2-MOE nucleotides, LNA nucleotides, and contains a mix In a preferred embodiment, one, two, three, four, or more nucleotides in the 3' overhang present in the sense and/or antisense strand of the siRNA are, for example, 2'OMe-guanosine nucleotides, 2'OMe- 2'OMe nucleotides such as uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof (eg, 2'OMe purine and/or pyrimidine nucleotides).

A siRNA is at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression. ) may be included. A cocktail of siRNAs can include sequences directed to the same region or domain (eg, a "hot spot") and/or to different regions or domains of one or more target genes. In certain instances, modified siRNAs that silence one or more (eg, at least two, three, four, five, six, seven, eight, nine, ten, or more) target gene expression are present in the cocktail. In certain other examples, unmodified siRNA sequences that silence one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) target gene expression are present in the cocktail. do.

Preferably, siRNA is chemically synthesized. siRNAs can also be generated by cleavage of longer dsRNAs (eg, dsRNAs greater than about 25 nucleotides in length) using E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA. Preferably, the dsRNA is at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be 1000, 1500, 2000, 5000 nucleotides in length or longer. A dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain instances, siRNAs can be encoded by a plasmid (eg, transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

The mRNA is preferably mono-, bi-, or multicistronic, as defined herein. Coding sequences within bi- or multicistronic mRNAs preferably encode individual peptides or proteins or fragments or variants thereof as defined herein. Preferably, coding sequences encoding two or more peptides or proteins may be separated from the bi- or multicistronic mRNA by at least one IRES (Internal Ribosome Entry Point), as defined below. Thus, the term “encoding two or more peptides or proteins” means that a bi- or multicistronic mRNA can contain, for example, at least 2, 3, 4, 5, 6 or more (within the definitions provided herein). preferably different) peptides or proteins or fragments or variants thereof, but is not limited thereto. More preferably, but not limited to, the bi- or multicistronic mRNA is at least 2, 3, 4, 5, 6 or more, for example as defined herein (preferably different) peptides or proteins or fragments or variants thereof as defined herein. In this context, the so-called IRES (Internal Ribosome Entry Point) sequence, as defined above, may serve as the sole ribosome binding site, but encode several peptides or proteins, as defined above, that are translated by the ribosome independently of each other. However, it may also serve to provide bi- or multicistronic mRNA. Examples of IRES sequences that may be used in accordance with the present invention are picornavirus (eg FMDV), pestivirus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classic swine fever virus (CSFV), mouse corneal leukoplakia virus (MLV), monkey immunodeficiency virus (SIV) or cricket paralysis virus (CrPV).

In the present invention, the cationic lipid refers to a compound of Formula 1 or a salt thereof:

[Formula 1]

'Hydrophobic lipid' refers to compounds having non-polar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

Nucleic acids present in the lipid-nucleic acid particles according to the present invention include nucleic acids in any known form. A nucleic acid as used herein may be single-stranded DNA or RNA, or double-stranded DNA or RNA, or a DNA-RNA hybrid. Examples of double-stranded DNA are described herein and include, for example, structural genes, genes comprising control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA are described herein and include, for example, siRNA and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triplex forming oligonucleotides.

Nucleic acids can generally be of various lengths depending on the particular form of the nucleic acid. For example, in certain embodiments, a plasmid or gene may be between about 1,000 and about 100,000 nucleotide residues in length. In certain embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In various related embodiments, the single-stranded, double-stranded, and triple-stranded oligonucleotides are about 10 to about 60 nucleotides in length, about 15 to about 60 nucleotides in length, about 20 to about 50 nucleotides in length, and about 15 to about 60 nucleotides in length. 30 nucleotides, or from about 20 to about 30 nucleotides in length.

In certain embodiments, an oligonucleotide (or strand thereof) of the invention specifically hybridizes to or is complementary to a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity such that stable and specific binding occurs between a DNA or RNA target and an oligonucleotide. It is understood that oligonucleotides need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. In a preferred embodiment, the oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence would interfere with the normal functioning of the target sequence, resulting in loss of usefulness or expression therefrom, and conditions under which specific binding is desired under physiological conditions, i.e., in the case of an in vivo assay or therapeutic treatment, or under the conditions under which the assay is performed, in the case of an in vitro assay, to a sufficient extent to prevent non-specific binding of the oligonucleotide to a non-target sequence. There is complementarity. Thus, an oligonucleotide may contain 1, 2, 3 or more base substitutions compared to the region of the gene or mRNA sequence that it targets or specifically hybridizes to.

In the present invention, the PEG-C-DMA has the following structure.

where n is selected such that the resulting polymer chain has a molecular weight of about 1000 to about 3000. In another embodiment, n is selected such that the resulting polymer chain has a molecular weight of about 2000. PEG-C-DMA is prepared as described in Heyes et al, Synthesis and Characterization of Novel Poly (Ethylene Glycol)-lipid Conjugates Suitable for use in Drug Delivery," Journal of Controlled Release, 2006, and U.S. Patent No. 8,936,942 Those skilled in the art will understand that the concentration of PEG-C-DMA can vary depending on the rate at which nucleic acid-lipid particles become soluble. For example, the rate at which nucleic acid-lipid particles become soluble is , for example by changing the molecular weight of PEG.

Examples of cholesterol derivatives are cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. including but not limited to

In the present invention, DSPC means distearoylphosphatidylcholine.

In certain embodiments, the present invention provides a method of continuous mixing, e.g., providing an aqueous solution comprising a nucleic acid to a first reservoir, providing an organic lipid solution to a second reservoir, and mixing the organic lipid solution with the aqueous solution A process comprising mixing an aqueous solution with an organic lipid solution to substantially immediately produce liposomes encapsulating a nucleic acid (eg, interfering RNA or mRNA). This process and apparatus for carrying out this process are described in detail in US Patent Publication No. 20040142025, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

Continuous introduction of the lipid and buffer solution into a mixing environment, such as a mixing chamber, results in serial dilution of the lipid solution with the buffer solution, producing liposomes substantially immediately upon mixing. As used herein, the phrase “serial dilution of a lipid solution with a buffer solution” (and variants) generally means that the lipid solution is diluted rapidly enough during the hydration process with sufficient force to cause vesicle formation. By mixing an aqueous solution containing nucleic acids with an organic lipid solution, the organic lipid solution undergoes serial stepwise dilution in the presence of a buffer solution (i.e., aqueous solution) to produce nucleic acid-lipid particles.

LNPs formed using continuous mixing methods typically have a wavelength of 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, or about 70 nm to about 90 nm. has a size of nm. The particles thus formed are not agglomerated and are optionally sized to obtain a uniform particle size.

The present invention provides an LNP produced through a direct dilution process in which a third reservoir containing a dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the liposomal solution formed in the first mixing zone is immediately and directly mixed with the dilution buffer in the second mixing zone. In a preferred embodiment, the second mixing zone comprises T-connectors arranged so that the liposomal solution and dilution buffer flows meet as opposing 180° flows; A connector providing a shallower angle, for example from about 27° to about 180°, may be used. A pump mechanism delivers a controllable buffer flow to the second mixing zone. In one aspect, the flow rate of the dilution buffer provided to the second mixing zone is controlled to be substantially the same as the flow rate of the liposome solution introduced from the first mixing zone. This embodiment advantageously allows better control of the flow of the dilution buffer mixed with the liposomal solution in the second mixing zone, and thus the concentration of the liposomal solution in the buffer throughout the second mixing process. Control of such dilution buffer flow rate advantageously allows small particle size formation at reduced concentrations.

The process and apparatus for performing this direct dilution process are described in detail in US Patent Publication No. 20070042031, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

LNPs formed using the direct dilution process typically have a wavelength of 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, or about 70 nm to about 90 nm. has a size of nm. The particles thus formed are not agglomerated and are optionally sized to obtain a uniform particle size.

If desired, lipid particles (eg, LNPs) of the invention may be sized by any method available for liposome sizing. Sizing can be performed to obtain a desired size range and relatively narrow particle size distribution. Several techniques are available for sizing the particles to the desired size. One sizing method used for liposomes and equally applicable to present particles is described in US Pat. No. 4,737,323, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Sonication of particle suspensions by bath or probe sonication results in gradual size reduction to particles less than about 50 nm in size. Homogenization is another method that relies on shear energy to break larger particles into smaller ones. In a typical homogenization procedure, the particles are recycled through a standard emulsion homogenizer until a selected particle size, typically about 60 to about 80 nm, is observed. In either method, the particle size distribution can be monitored by conventional laser-beam particle size identification, or QELS.

Extrusion of particles through small pore polycarbonate membranes or asymmetric ceramic membranes is also an effective way to reduce particle size to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. Particles can be extruded through successively smaller pore membranes to achieve progressive size reduction.

In one aspect of the present invention, the peptide is conjugated to form a complex with a lipid nanoparticle comprising:

(i) a cationic lipid having Formula 2:

[Formula 2]

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

L ¹ and L ² are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond;

R ^1a and R ^1b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^1a is H or C ₁ -C ₁₂ alkyl, and R ^1b is the carbon atom to which it is attached. together with adjacent R ^1b and the carbon atom to which it is attached to form a carbon-carbon double bond;

R ^2a and R ^2b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^2a is H or C ₁ -C ₁₂ alkyl, and R ^2b is the carbon atom to which it is attached. together with adjacent R ^2b and the carbon atom to which it is attached to form a carbon-carbon double bond;

R ^3a and R ^3b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^3a is H or C ₁ -C ₁₂ alkyl, and R ^3b is the carbon atom to which it is attached. together with adjacent R ^3b and the carbon atom to which it is attached to form a carbon-carbon double bond;

R ^4a and R ^4b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^4a is H or C ₁ -C ₁₂ alkyl, and R ^4b is the carbon atom to which it is attached. together with adjacent R ^4b and the carbon atom to which it is attached to form a carbon-carbon double bond;

R ⁵ and R ⁶ are each independently methyl or cycloalkyl;

R ⁷ , at each occurrence, is independently H or C ₁ -C ₁₂ alkyl;

R ⁸ and R ⁹ are each independently C ₁ -C ₁₂ alkyl; R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle containing one nitrogen atom;

a and d are each independently an integer of 0 to 24;

b and c are each independently an integer from 1 to 24;

e is 1 or 2); and

(ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

A complex comprising a.

In some embodiments of Formula 2, at least one of R ^1a , R ^2a , R ^3a or R ^4a is C ₁ -C ₁₂ alkyl, or at least one of L ¹ or L ² is -O(C=0)- or - (C=O)O-. In other embodiments, R ^1a and R ^1b are not isopropyl when a is 6 or n-butyl when a is 8.

In some additional embodiments of Formula 2, at least one of R ^1a , R ^2a , R ^3a or R ^4a is C ₁ -C ₁₂ alkyl, or at least one of L ¹ or L ² is -O(C=O)- or -(C=O)O-; R ^1a and R ^1b are not isopropyl when a is 6 or n-butyl when a is 8.

In another embodiment of Formula @, R ⁸ and R ⁹ are each independently unsubstituted C ₁ -C ₁₂ alkyl; R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle containing one nitrogen atom.

In some embodiments of Formula 2, any one of L ¹ or L ² can be -O(C=O)- or a carbon-carbon double bond. L ¹ and L ² may each be -O(C=O)-, or each may be a carbon-carbon double bond.

In some embodiments of Formula 2, one of L ¹ or L ² is -O(C=O)-. In another embodiment, both L ¹ and L ² are -O(C=0)-.

In some embodiments of Formula 2, one of L ¹ or L ² is -(C=0)0-. In another embodiment, both L ¹ and L ² are -(C=0)0-.

In some other embodiments of Formula 2, either L ¹ or L ² is a carbon-carbon double bond. In other embodiments, both L ¹ and L ² are carbon-carbon double bonds.

In yet another embodiment of Formula 2, one of L ¹ or L ² is -O(C=O)- and the other of L ¹ or L ² is -(C=O)O-. In more embodiments, one of L ¹ or L ² is -O(C=O)- and the other of L ¹ or L ² is a carbon-carbon double bond. Still in more embodiments, one of L ¹ or L ² is -(C=0)O- and the other of L ¹ or L ² is a carbon-carbon double bond.

As used throughout the specification, a “carbon-carbon” double bond is understood to refer to one of the following structures:

or

In this case, R ^a and R ^b are, in each case, independently H or a substituent. For example, in some embodiments, R ^a and R ^b , at each occurrence, independently are H, C ₁ -C ₁₂ alkyl or cycloalkyl, such as H or C ₁ -C ₁₂ alkyl.

In another embodiment, the lipid compound of Formula 2 has structure 2a:

[2a]

In another embodiment, the lipid compound of Formula 2 has structure 2b:

[2b]

In another embodiment, the lipid compound of formula 2 has structure 2c:

[2c]

In some embodiments of the lipid compound of Formula 2, a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In another embodiment, a, b, c and d are each independently an integer from 8 to 12 or from 5 to 9. In some specific embodiments, a is zero. In some embodiments, a is 1. In another embodiment, a is 2. In more embodiments, a is 3. In another embodiment, a is 4. In some embodiments, a is 5. In another embodiment, a is 6. In more embodiments, a is 7. In another embodiment, a is 8. In some embodiments, a is 9. In another embodiment, a is 10. In more embodiments, a is 11. In another embodiment, a is 12. In some embodiments, a is 13. In another embodiment, a is 14. In more embodiments, a is 15. In another embodiment, a is 16.

In some other embodiments of Formula 2, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In another embodiment, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In another embodiment, b is 8. In some embodiments, b is 9. In other embodiments, b is 10.

In more embodiments, b is 11. In another embodiment, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In another embodiment, b is 16.

In some more embodiments of Formula 2, c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In another embodiment, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In another embodiment, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In another embodiment, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In another embodiment, c is 16.

In some specific other embodiments of Formula 2, d is 0. In some embodiments, d is 1. In another embodiment, d is 2. In more embodiments, d is 3. In another embodiment, d is 4. In some embodiments, d is 5. In another embodiment, d is 6. In more embodiments, d is 7. In another embodiment, d is 8. In some embodiments, d is 9. In another embodiment, d is 10. In more embodiments, d is 11. In another embodiment, d is 12. In some embodiments, d is 13. In another embodiment, d is 14. In more embodiments, d is 15. In another embodiment, d is 16.

In some other various embodiments of Formula 2, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are equal and b and c are equal.

The sum of a and b and the sum of c and d in Formula 2 are factors that can be varied to obtain a lipid of Formula 2 having the desired properties. In one implementation, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In a further embodiment, the sum of a and b and the sum of c and d are equal. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which can range from 14 to 24. Also, in a further embodiment, a. b, c and d are selected so that the sum of a and b and the sum of c and d is 12 or more.

In some embodiments of Formula 2, e is 1. In another embodiment, e is 2.

Substituents in R ^1a , R ^2a , R ^3a and R ^4a in Formula 2 are not particularly limited. In some embodiments, R ^1a , R ^2a , R ^3a and R ^4a are H at each occurrence. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₁₂ alkyl. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₈ alkyl. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₆ alkyl. In some of the foregoing embodiments, C ₁ -C ₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl. In some embodiments of Formula 2, R ^1a , R ^2a , R ^3a and R ^4a are at each occurrence C ₁ -C ₁₂ alkyl.

In a further embodiment of Formula 2, at least one of R ^1a , R ^2a , R ^3a and R ^4a is H, or R ^1a , R ^2a , R ^3a and R ^4a are in each case H.

In some embodiments of Formula 2, R ^1b is taken together with the carbon atom to which it is attached, together with adjacent R ^1b and the carbon atom to which it is attached to form a carbon-carbon double bond. In some embodiments of the foregoing, R ^4b is taken together with the carbon atom to which it is attached, together with adjacent R4b and the carbon atom to which it is attached to form a carbon-carbon double bond.

Substituents for R ⁵ and R ⁶ in Formula 2 are not particularly limited in the above embodiments. In some embodiments, one or both of R ⁵ or R ⁶ is methyl. In some other embodiments, one or both of R ⁵ or R ⁶ is cycloalkyl, such as cyclohexyl. In these embodiments, cycloalkyls can be substituted or unsubstituted. In some other embodiments, cycloalkyl is substituted with C ₁ -C ₁₂ alkyl, for example tert-butyl.

Substituents in R ⁷ are not particularly limited in the above-described embodiments of Formula 2. In some embodiments, at least one R ⁷ is H. In some other embodiments, R ⁷ is H at each occurrence. In some other embodiments, R ⁷ is C ₁ -C ₁₂ alkyl.

In some other embodiments of Formula 2 above, one of R ⁸ or R ⁹ is methyl. In other embodiments, both R ⁸ and R ⁹ are methyl.

In some different embodiments of Formula 2, R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle. In some embodiments of the foregoing, R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5-membered heterocycle, such as a pyrrolidinyl ring.

In various different embodiments, the lipid of Formula 2 has one of the structures set forth in Table 1 below.

In some embodiments, the LNP comprises a lipid of Formula 2, an mRNA compound as defined herein, and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of formula 2 is compound 2-5. In some embodiments, the lipid of formula 2 is compound 2-6.

In one aspect of the present invention, the peptide is conjugated to form a complex with a lipid nanoparticle comprising:

(i) a cationic lipid having Formula 3:

[Formula 3]

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

L ¹ and L ² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -SS- , -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, -NR ^a C(=O)NRa-, - OC(=O)NR ^a -, -NR ^a C(=O)O-, or a direct bond;

G ¹ is C ₁ -C ₂ alkylene, -(C=O)- , -O(C=O)-, -SC(=O)-, -NR ^a C(=O)- or a direct bond;

G ² is -C(=0)- , -(C=0)0-, -C(=0)S-, -C(=0)NR ^a - or a direct bond;

G ³ is C ₁ -C ₆ alkylene;

R ^a is H or C ₁ -C ₁₂ alkyl;

R ^1a and R ^1b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^1a is H or C ₁ -C ₁₂ alkyl, R ^1b taken together with the carbon atom to which it is attached, together with adjacent R ^1b and the carbon atom to which it is attached, form a carbon-carbon double bond;

R ^2a and R ^2b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^2a is H or C ₁ -C ₁₂ alkyl, and R ^2b is taken together with the carbon atom to which it is attached, together with adjacent R ^2b and the carbon atom to which it is attached, to form a carbon-carbon double bond;

R ^3a and R ^3b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^3a is H or C ₁ -C ₁₂ alkyl, and R ^3b is taken together with the carbon atom to which it is attached, together with adjacent R ^3b and the carbon atom to which it is attached, to form a carbon-carbon double bond;

R ^4a and R ^4b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^4a is H or C ₁ -C ₁₂ alkyl, and R ^4b is taken together with the carbon atom to which it is attached, together with adjacent R ^4b and the carbon atom to which it is attached to form a carbon-carbon double bond;

R ⁵ and R ⁶ are each independently H or methyl;

R ⁷ is C ₄ -C ₂₀ alkyl;

R ⁸ and R ⁹ are each independently C ₁ -C ₁₂ alkyl; R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle;

a, b, c and d are each independently an integer from 1 to 24;

x is 0, 1 or 2; and

(ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

A complex comprising a.

In some embodiments of Formula 3, L ¹ and L ² are each independently -O(C=O)-, -(C=O)O-, or a direct bond. In another embodiment, G ¹ and G ² are each independently -(C=0)- or a direct bond. In some different embodiments, L ¹ and L ² are each independently -O(C=0)-, -(C=0)0-, or a direct bond; G ¹ and G ² are each independently -(C=O)- or a direct bond.

In some different embodiments of Formula 3, L ¹ and L ² are each independently -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S- , -SC(=O)-, -NRa-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa, -OC(=O)NRa-, -NRaC( =O)O-, -NRaS(O)xNRa-, -NRaS(O)x- or -S(O)xNRa-.

In another of the aforementioned embodiments of Formula 3, the lipid compound has one of the following structures 3A or 3B:

[3A] or

[3B]

In some embodiments of Formula 3, the lipid compound has structure 3A. In another embodiment, the lipid compound has structure 3B.

In any of the preceding embodiments of Formula 3, one of L ¹ or L ² is -O(C=O)-. For example, in some embodiments, each of L ¹ and L ² is -O(C=0)-.

In some different embodiments of Formula 3, one of L ¹ or L ² is -(C=0)0-. For example, in some embodiments, each of L ¹ and L ² is -(C=0)0-.

In a different embodiment of Formula 3, either L ¹ or L ² is a direct bond. As used herein, “direct bond” means the absence of the corresponding group (eg, L ¹ or L ² ). For example, in some embodiments, each of L ¹ and L ² is a direct bond.

In other different embodiments of Formula 3, for at least one occurrence of R ^1a and R ^1b , R ^1a is H or C ₁ -C ₁₂ alkyl, and R ^1b together with the carbon atom to which it is attached, adjacent R ^1b and Used together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond.

In still other different embodiments of Formula 3, for at least one occurrence of R ^4a and R ^4b , R ^4a is H or C ₁ -C ₁₂ alkyl, and R ^4b together with the carbon atom to which it is attached, adjacent R ^4b and the carbon atom to which it is bonded forms a carbon-carbon double bond.

In more embodiments of Formula 3, for at least one occurrence of R ^2a and R ^2b , R ^2a is H or C ₁ -C ₁₂ alkyl, and R ^2b together with the carbon atom to which it is attached, adjacent R ^2b and Used together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond.

In other different embodiments of Formula 3, for at least one occurrence of R ^3a and R ^3b , R ^3a is H or C ₁ -C ₁₂ alkyl, R ^3b together with the carbon atom to which it is attached, adjacent R ^3b and Used together with the carbon atom to which it is bonded, it forms a carbon-carbon double bond.

In various other embodiments of Formula 3, the lipid compound has either structure 3C or 3D:

[3C] or

[3D]

(In the formula, e, f, g and h are each independently an integer of 1 to 12).

In some embodiments of Formula 3, the lipid compound has structure 3C. In another embodiment, the lipid compound has a 3D structure.

In various embodiments of structure 3C or 3D, e, f, g and h are each independently an integer from 4 to 10.

In some embodiments of Formula 3, a, b, c, and d are each independently an integer from 2 to 12 or an integer from 4 to 12.

In another embodiment, a, b, c and d are each independently an integer from 8 to 12 or from 5 to 9. In some specific embodiments, a is zero. In some embodiments, a is 1. In another embodiment, a is 2. In more embodiments, a is 3. In another embodiment, a is 4. In some embodiments, a is 5. In another embodiment, a is 6. In more embodiments, a is 7. In another embodiment, a is 8. In some embodiments, a is 9. In another embodiment, a is 10. In more embodiments, a is 11. In another embodiment, a is 12. In some embodiments, a is 13. In another embodiment, a is 14. In more embodiments, a is 15. In another embodiment, a is 16.

In some embodiments of Formula 3, b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In another embodiment, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In another embodiment, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In another embodiment, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In another embodiment, b is 16.

In some embodiments of Formula 3, c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In another embodiment, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In another embodiment, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In another embodiment, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In another embodiment, c is 16.

In some specific embodiments of Formula 3, d is 0. In some embodiments, d is 1. In another embodiment, d is 2. In more embodiments, d is 3. In another embodiment, d is 4. In some embodiments, d is 5. In another embodiment, d is 6. In more embodiments, d is 7. In another embodiment, d is 8. In some embodiments, d is 9. In another embodiment, d is 10. In more embodiments, d is 11. In another embodiment, d is 12. In some embodiments, d is 13. In another embodiment, d is 14. In more embodiments, d is 15. In another embodiment, d is 16.

In some embodiments of Formula 3, e is 1. In another embodiment, e is 2. In more embodiments, e is 3. In another embodiment, e is 4. In some embodiments, e is 5. In another embodiment, e is 6. In more embodiments, e is 7. In another embodiment, e is 8. In some embodiments, e is 9. In another embodiment, e is 10. In more embodiments, e is 11. In another embodiment, e is 12.

In some embodiments of Formula 3, f is 1. In another embodiment, f is 2. In more embodiments, f is 3. In another embodiment, f is 4. In some embodiments, f is 5. In another embodiment, f is 6. In more embodiments, f is 7. In another embodiment, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In another embodiment, f is 12.

In some embodiments of Formula 3, g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In another embodiment, g is 4. In some embodiments, g is 5. In another embodiment, g is 6. In more embodiments, g is 7. In another embodiment, g is 8. In some embodiments, g is 9. In another embodiment, g is 10. In more embodiments, g is 11. In another embodiment, g is 12.

In some embodiments of Formula 3, h is 1. In another embodiment, e is 2. In more embodiments, h is 3. In another embodiment, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In another embodiment, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In another embodiment, h is 12.

In some other various embodiments of Formula 3, a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are equal and b and c are equal.

The sum of a and b and the sum of c and d in Formula 3 are factors that can be varied in order to obtain a lipid having the desired properties. In one implementation, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In a further embodiment, the sum of a and b and the sum of c and d are equal. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which can range from 14 to 24. Also, in a further embodiment, a. b, c and d are selected so that the sum of a and b and the sum of c and d is 12 or more.

Substituents for R ^1a , R ^2a , R ^3a and R ^4a in Formula 3 are not particularly limited. In some embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is H. In some embodiments, R ^1a , R ^2a , R ^3a and R ^4a are H at each occurrence. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₁₂ alkyl. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₈ alkyl. In some other embodiments, at least one of R ^1a , R ^2a , R ^3a and R ^4a is C ₁ -C ₆ alkyl. In some of the foregoing embodiments, C ₁ -C ₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In some embodiments of Formula 3, R ^1a , R ^2a , R ^3a and R ^4a at each occurrence are C ₁ -C ₁₂ alkyl.

In a further embodiment of Formula 3, at least one of R ^1a , R ^2a , R ^3a and R ^4a is H, or R ^1a , R ^2a , R ^3a and R ^4a are in each case H.

In some embodiments of Formula 3, R ^1b is taken together with the carbon atom to which it is attached, together with adjacent R ^1b and the carbon atom to which it is attached to form a carbon-carbon double bond. In some embodiments of the foregoing, R ^4b is taken together with the carbon atom to which it is attached, together with adjacent R ^4b and the carbon atom to which it is attached to form a carbon-carbon double bond.

Substituents for R ⁵ and R ⁶ in Formula 3 are not particularly limited in the above embodiments. In some embodiments, one of R ⁵ or R ⁶ is methyl. In other embodiments, each of R ⁵ or R ⁶ is methyl.

Substituents in R ⁷ of Formula 3 are not particularly limited in the above embodiments. In some embodiments, R ⁷ is C ₆ -C ₁₆ alkyl. In some other embodiments, R ⁷ is C ₆ -C ₉ alkyl. In some of these embodiments, R7 is -(C=0)ORb, -O(C=0)Rb, -C(=0)Rb, -ORb, -S(0)xRb, -S-SRb, - C(=O)SRb, -SC(=O)Rb, -NRaRb, -NRaC(=O)Rb, -C(=O)NRaRb, -NRaC(=O)NRaRb, -OC(=O)NRaRb, Substituted with -NRaC(=O)ORb, -NRaS(O)xNRaRb, -NRaS(O)xRb or -S(O)xNRaRb,

In this case, R ^a is H or C ₁ -C ₁₂ alkyl; R ^b is C ₁ -C ₁₅ alkyl; x is 0, 1 or 2; For example, in some embodiments, R ⁷ is substituted with -(C=0)ORb or -0(C=0)Rb.

In various of the aforementioned embodiments of Formula 3, R ^b is a branched C ₁ -C ₁₅ alkyl. For example, in some embodiments, R ^b has one of the following structures:

In some other embodiments of Formula 3 above, one of R ⁸ or R ⁹ is methyl. In other embodiments, both R8 and R9 are methyl.

In some different embodiments of Formula 3, R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle. In some embodiments of the foregoing, R8 and R9 together with the nitrogen atom to which they are attached form a 5-membered heterocycle, such as a pyrrolidinyl ring. In some of the different embodiments described above, R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 6-membered heterocycle, such as a piperazinyl ring.

In another embodiment of the aforementioned lipid of Formula 3, G ³ is C ₂ -C ₄ alkylene, eg C ₃ alkylene.

In a variety of different embodiments, the lipid compound has one of the structures set forth in Table 2 below.

In some embodiments, the LNP comprises a lipid of Formula 3, an mRNA compound as described above, and one or more excipients selected from neutral lipids, steroids, and pegylated lipids. In some embodiments, the lipid of formula 3 is compound 3-9. In some embodiments, the lipid of formula 3 is compound 3-10. In some embodiments, the lipid of formula 3 is compound 3-11. In some embodiments, the lipid of formula 3 is compound 3-12. In some embodiments, the lipid of formula 3 is compound 3-32.

In one aspect of the present invention, the peptide is conjugated to form a complex with a lipid nanoparticle comprising:

(i) a cationic lipid having Formula 4:

[Formula 4]

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

L ¹ or L ² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -SS- , -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, -NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O-;

Preferably L ¹ or L ² is -O(C=O)- or -(C=O)O-;

G ¹ and G ² are each independently unsubstituted C ₁ -C ₁₂ alkylene or C ₁ -C ₁₂ alkenylene;

G ³ is C ₁ -C ₂₄ alkylene, C ₁ -C ₂₄ alkenylene, C ₃ -C ₈ cycloalkylene, or C ₃ -C ₈ cycloalkenylene;

R ^a is H or C ₁ -C ₁₂ alkyl;

R ¹ and R ² are each independently C ₆ -C ₂₄ alkyl or C ₆ -C ₂₄ alkenyl;

R ³ is H, OR ⁵ , CN, -C(=0)OR ⁴ , -OC(=0)R ⁴ or -NR ⁵ C(=0)R ⁴ ;

R ⁴ is C ₁ -C ₁₂ alkyl;

R ⁵ is H or C ₁ -C ₆ alkyl;

x is 0, 1 or 2; and

(ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

A complex comprising a.

In some embodiments of the aforementioned embodiments of Formula 4 above, the lipid has one of the following structural Formulas 4A or 4B:

[Formula 4A] or

[Formula 4B]

where A is a 3 to 8 membered cycloalkyl or cycloalkylene ring;

R ⁶ , at each occurrence, is independently H, OH, or C ₁ -C ₂₄ alkyl;

n is an integer ranging from 1 to 15.

In some of the aforementioned embodiments of Formula 4, the lipid has Formula 4A, and in other embodiments the lipid has Formula 4B.

In another embodiment of Formula 4, the lipid has either Formula 4C or Formula 4D:

[Formula 4C] or

[Formula 4D]

In this case, y and z are each independently an integer ranging from 1 to 12.

In any of the preceding embodiments of Formula 4, one of L ¹ or L ² is -O(C=O)-. For example, in some embodiments, each of L ¹ and L ² is -O(C=0)-. In some different embodiments of any of the foregoing, L1 and L2 are each independently -(C=0)0- or -0(C=0)-. For example, in some embodiments, each of L ¹ and L ² is -(C=0)0-.

In some different embodiments of Formula 4, the lipid has one of the following Formulas 4E or 4F:

[Formula 4E] or

[Formula 4F]

In some embodiments of the foregoing embodiments of Formula 4, the lipid has one of the following structural Formula 4G, Formula 4H, Formula 4I, or Formula 4J:

[Formula 4G];

[Formula 4H];

[Formula 4I] or

[Formula 4J]

In some embodiments of the aforementioned embodiments of Formula 4, n is an integer ranging from 2 to 12, such as from 2 to 8 or from 2 to 4. For example, in some embodiments n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the aforementioned embodiments of Formula 4, y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or 4 to 6.

In some of the aforementioned embodiments of Formula 4, R ⁶ is H. In other of the foregoing embodiments, R ⁶ is C ₁ -C ₂₄ alkyl. In other embodiments, R ⁶ is OH.

In some embodiments of Formula 4, G ³ is unsubstituted. In other embodiments, G ³ is unsubstituted. In various different embodiments, G ³ is linear C ₁ -C ₂₄ alkylene or linear C ₁ -C ₂₄ alkenylene.

In some other aforementioned embodiments of Formula 4, either R ¹ or R ² , or both, is C ₆ -C ₂₄ alkenyl. For example, in some embodiments, each of R ¹ and R ² independently has the structure:

wherein R ^7a and R ^7b , at each occurrence, are independently H or C ₁ -C ₁₂ alkyl;

a is an integer from 2 to 12;

R ^7a , R ^7b and a are each selected such that R ¹ and R ² each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

In some embodiments of the aforementioned embodiments of Formula 4, at least one instance of R ^7a is H. For example, in some embodiments, R ^7a is H at each occurrence. In other different of the foregoing embodiments, at least one instance of R ^7b is C ₁ -C ₈ alkyl. For example, in some embodiments, C ₁ -C ₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In a different embodiment of Formula 4, R ¹ or R ² , or both have one of the following structures:

In some of the aforementioned embodiments of Formula 4, R ³ is OH, CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ or -NHC(=O)R ⁴ . In some embodiments, R ⁴ is methyl or ethyl.

In various different embodiments, the cationic lipid of Formula 4 has one of the structures set forth in Table 3 below.

In some embodiments, the LNP comprises a lipid of Formula 4, an mRNA compound as described herein, and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of formula 4 is compound 4-3. In some embodiments, the lipid of formula 4 is compounds 4-7.

Within the context of the present invention, LNP-4-3 means a lipid nanoparticle as defined herein comprising a cationic lipid compound 4-3 according to the table above. Other lipid nanoparticles are referred to in a similar fashion.

In some embodiments, the cationic lipid of Formula 2, 3, or 4 is present in the LNP in an amount from about 30 to about 95 mole percent relative to the total lipid content of the LNP. When two or more cationic lipids are incorporated into an LNP, such percentages apply to the combined cationic lipids. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mole percent. In one embodiment, each cationic lipid is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or in an amount of about 40 to about 60 mole percent, such as 60 mole percent.

In some embodiments of the invention, the LNP comprises a combination or mixture of any of the lipids described above.

In one of the preferred embodiments, the lipid nanoparticle comprises a cationic lipid selected from the group:

In a preferred embodiment, the lipid nanoparticle is 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 , 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm in average diameter, and is substantially non-toxic. As mentioned, the average diameter may correspond to a z-average value determined by dynamic light scattering.

In another preferred embodiment of the present invention, the lipid nanoparticle has a range of about 50 nm to about 300 nm, or about 60 nm to about 250 nm, about 60 nm to about 150 nm, or about 60 nm to about 120 nm, respectively. has a hydrodynamic diameter.

In some embodiments, mRNA resists degradation by nucleases in aqueous solutions when present in lipid nanoparticles.

The total amount of mRNA in lipid nanoparticles varies and can be defined according to the mRNA w/w ratio to total lipid. In one embodiment of the invention, the mRNA to total lipid ratio is less than 0.06 w/w, preferably between 0.03 and 0.04 w/w.

In some embodiments, the LNP comprises a lipid of formula 2, 3 or 4, an mRNA compound as defined above, a neutral lipid, a steroid. In some embodiments, the lipid of formula 2 is compound 2-6, or the lipid of formula 4 is compound 4-3, the neutral lipid is DSPC, and the steroid is cholesterol.

In some embodiments, the LNP comprises one or more targeting moieties capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor found on the cell surface.

In some embodiments, an LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells and induce internalization of the LNP. For example, in one embodiment, the one or more internalization mains bind to a receptor found on the cell surface and induce receptor-mediated uptake of the LNP. In some embodiments, the LNP may bind to a biomolecule in vivo, and in vivo the LNP-bound biomolecule may be recognized by a cell surface receptor to induce internalization. For example, in one embodiment, LNP binds systemic ApoE, which results in uptake of the LNP and associated cargo.

The following reaction schemes illustrate methods for preparing lipids of Formula 2, 3 or 4.

General Scheme 1

Embodiments of Formula 2 lipids (eg, Compounds A-5) can be prepared according to general Scheme 1 (“Method A”), wherein R is saturated or unsaturated C ₁ -C ₂₄ alkyl or saturated or unsaturated cycloalkyl And, m is 0 or 1, n is an integer from 1 to 24. Referring to General Scheme 1, compounds of Structure A-1 may be purchased from commercial sources or may be prepared according to methods familiar to those skilled in the art. A mixture of A-1, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine) and N,N-dimethyldiamine A-4 to produce A-5 after any necessary workup and or purification steps. Heat at sufficient temperature and time.

General Scheme 2

Other embodiments of compounds of Formula 2 (eg, Compound B-5) can be prepared according to general Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C ₁ -C ₂₄ alkyl or saturated or unsaturated Cycloalkyl, m is 0 or 1, n is an integer from 1 to 24. As shown in General Scheme 2, compounds of structure B-1 can be purchased from commercial sources or can be prepared according to methods familiar to those skilled in the art. A solution of B-1 (1 equiv.) is treated with the acid chloride B-2 (1 equiv.) and a base (eg triethylamine). The crude product is treated with an oxidizing agent (eg pyridinium chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, acid (eg acetic acid) and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (eg sodium triacetoxyborohydride) to allow for any necessary work-up and / or after purification, B-5 is obtained.

Although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbons, starting materials containing carbon-carbon double bonds can also be utilized in the preparation of compounds containing carbon-carbon double bonds. Be careful.

General Scheme 3

Different embodiments of lipids of Formula 2 (e.g., compounds C-7 or C9) can be prepared according to general Scheme 3 ("Method C"), where R is a saturated or unsaturated C1-C24 alkyl or a saturated or unsaturated Cycloalkyl, m is 0 or 1, n is an integer from 1 to 24. Referring to General Scheme 3, compounds of structure C-1 can be purchased from commercial sources or can be prepared according to methods familiar to those skilled in the art.

General Scheme 4

Embodiments of compounds of Formula 3 (eg, compounds D-5 and D-7) can be prepared according to general Scheme 4 (“Method D”), wherein R ^1a , R ^1b , R ^2a , R ^2b , R ^3a , R ^3b , R ^{4a ,} R 4b , R ⁵ , R ⁶ , R ⁸ , R ⁹ , L ¹ , L ² , G ¹ , G ² , G ³ , a, b, c and d As defined, R ^7' represents R ⁷ or C ₃ -C ₁₉ alkyl. Referring to General Scheme 1, compounds of structures D-1 and D-2 may be purchased from commercial sources or may be prepared according to methods familiar to those skilled in the art. Solutions of D-1 and D-2 are treated with a reducing agent (eg sodium triacetoxyborohydride) to obtain D-3 after any necessary work-up. A solution of D-3 and base (e.g., trimethylamine, DMAP) is treated with the acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary workup and/or purification. D-5 can be reduced to LiAlH4 D-6 to provide D-7 after any necessary workup and/or purification.

General Scheme 5

Embodiments of lipids of Formula 3 (eg, Compound E-5) can be prepared according to general Scheme 5 (“Method E”), wherein R ^1a , R ^1b , R ^2a , R ^2b , R ^3a , R ^3b , R ^4a , R ^4b , R ^{5 , R 6} , R ⁷ , R ⁸ , R ⁹ , L ¹ , L ² , G ³ , a, b, ^c and d are as defined herein. Referring to General Scheme 2, compounds of structures E-1 and E-2 may be purchased from commercial sources or may be prepared according to methods familiar to those skilled in the art. Heat the mixture of (excess) E-1, E-2 and a base (eg potassium carbonate) to obtain E-3 after any necessary work-up. A solution of E-3 and a base (e.g., trimethylamine, DMAP) is treated with the acyl chloride E-4 (or carboxylic acid and DCC) to yield E-5 after any necessary workup and/or purification.

General Scheme 6

Other embodiments of compounds of Formula 3 (eg, F-9) are prepared according to general Scheme 6 (Method F). As shown in general Scheme 6, a suitably protected ketone (F-1) is reacted with an amine F-2 under reductive amination conditions to give F-3. Acylation of F-3 with acid chloride F-4 yields acylation product F-5. Removal of the alcohol protecting group on F-5, followed by reaction with F-7 and/or F-8 and a suitable activating reagent (eg DCC) yields the desired compound F-9.

General Scheme 7

General Scheme 7 provides an exemplary method for the preparation of lipids of Formula 4 (Method G). G ¹ , G ³ , R ¹ and R ³ in General Reaction Scheme 7 are as defined herein for Formula 4, and G ^1′ refers to a homologue of G ¹ that is one carbon shorter. Compounds of structure G-1 are purchased or prepared according to methods known in the art. Reaction of G-1 with the diol G-2 under suitable condensation conditions (eg DCC) yields the ester/alcohol G-3, which can then be oxidized to the aldehyde G-4 (eg PCC). Reaction of amines G-5 and G-4 under reductive amination conditions produces lipids of Formula 4.

It should be noted that a variety of alternative strategies for the preparation of lipids of Formula 4 are available to those skilled in the art. For example, other lipids of Formula 4, wherein L ¹ and L ² are other than esters, can be prepared according to similar methods using suitable starting materials. General Scheme 6 also illustrates the preparation of lipids of Formula 4, wherein G ¹ and G ² are the same. However, this is not a required aspect of the present invention, and modifications to the above reaction scheme are possible to produce compounds having different G ¹ and G ² .

It will be appreciated by those skilled in the art that in the methods described herein, functional groups of intermediate compounds may need to be protected with suitable protecting groups. These functional groups include hydroxy, amino, mercapto and carboxylic acids. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (eg, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, adino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters. Protecting groups are known to those skilled in the art and may be added or removed according to general techniques as described herein. The use of protecting groups is known in the art. As will be appreciated by those skilled in the art, the protecting group may also be a polymeric resin such as Wang resin, Rink resin or 2-chlorotrityl-chloride resin.

In one aspect of the present invention, the peptide may be selected from the group consisting of SEQ ID NOs: 1 to 10.

According to one embodiment of the present invention, peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 10 show excellent cell membrane permeability compared to the permeability of conventional TAT proteins, and the higher the treatment concentration, the higher the intracellular permeability. confirmed to indicate

The present invention provides a composition for promoting cell permeation of a substance comprising a complex comprising a lipid nanoparticle and a cell penetrating peptide.

In the present invention, the composition may be used to deliver biologically active substances into living tissue or blood or promote cell permeation. The composition may be delivered through cells constituting biological tissues or cell-to-cell junctions, but the delivery method is not limited.

The biological tissue refers to one or more epithelial tissue, muscle tissue, nerve tissue, and connective tissue, and each organ may consist of one or more tissues, such as mucous membrane, skin, brain, lung, liver, kidney, spleen, lung, heart, and stomach. , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genital organ, muscle, blood, blood vessel, lymphatic vessel, lymph node, thymus, pancreas, adrenal gland, thyroid, parathyroid gland, larynx, tonsils, bronchi, and alveoli. However, it is not limited to the above example.

Cells constituting the biological tissue include epithelial cells, muscle cells, nerve cells, glandular cells, glial cells, germ cells, stem cells, mesenchymal cells, mesenchymal stem cells, osteoblasts, osteocytes, osteoblasts, osteoclasts, blood cells Cells, hematopoietic cells, lung cells, hepatocytes, fibroblasts, immune cells, endothelial cells, adipocytes, chondrocytes, etc. may be included, but are not limited to the above examples.

Epithelial cells include mucosal epithelial cells, hair follicle epithelial cells, digestive tract epithelial cells, respiratory epithelial cells, genital epithelial cells, urinary epithelial cells, and the like. Endothelial cells include vascular endothelial cells or lymphatic endothelial cells, but are not limited to the above examples.

As one aspect of the present invention, the composition may be for delivering biologically active substances through mucous membranes or skin, but is not limited to the above examples.

The composition of the present invention can be used for various purposes depending on the biologically active material included in the composition together with the complex. For example, the compositions of the present invention can be used to treat diseases in humans or animals. The type of the disease is not limited, but it may be a disease that requires high concentration drug delivery or improved drug delivery into cells, tissues, or blood, for example, treatment with a gene therapy drug or a protein therapy drug with a high molecular weight. It can be applied to various diseases for possible diseases or treatments that require a change in the route of administration. The disease includes, for example, breast cancer, liver cancer, brain cancer, prostate cancer, uterine cancer, ovarian cancer, stomach cancer, esophageal cancer, colon cancer, rectal cancer, thyroid cancer, blood cancer, skin cancer, cancer including lung cancer, diabetes, obesity, asthma, alopecia, and the like. It may be, but is not limited thereto. In addition, it can be used for research purposes to observe cell membrane permeability in vitro or intracellular delivery of drugs using the peptide through the cell membrane, and treatment of diseases treated with gene therapy drugs or high molecular weight protein therapy drugs such as cancer and diabetes can also be utilized. The peptide can be used in various molecules to diagnose contrast agents, diagnostic reagents and kits, and can also be used in health functional foods (health functional food compositions) and cosmetics (cosmetic compositions), but is not limited to the above examples.

In addition, the present invention provides a use of a material comprising the lipid nanoparticle and the peptide complex of the present invention for preparing a composition for promoting cell permeation.

In order to achieve another object of the present invention, the present invention promotes cell permeation comprising administering an effective amount of a composition containing as an active ingredient a substance containing the lipid nanoparticle and the peptide complex of the present invention to a subject in need thereof provides a way

The 'effective amount' of the present invention refers to an amount that exhibits an improvement or promotion effect on cell permeation rate when administered to an individual, and the 'individual' may be an animal, preferably a mammal, particularly an animal including a human, , cells, tissues, organs, etc. derived from animals. The subject may be a patient in need of the effect.

In this specification, the term "comprising" is used in the same meaning as "including" or "characterized by", and in the composition or method according to the present invention, specifically mentioned It does not exclude additional components or method steps not specified. Also, the term "consisting of" means excluding additional elements, steps or components not separately described. The term "essentially consisting of" means that in the scope of a composition or method, in addition to the described materials or steps, materials or steps that do not substantially affect the basic characteristics thereof may be included.

Therefore, the complex including the lipid nanoparticle of the present invention and the novel cell-penetrating peptide can effectively penetrate into living organisms such as cells, tissues, and blood.

1a to 1f show BEAS-2B cells or HeLa cells treated with the peptides of the present invention at various concentrations, 15 minutes (1a, 1b), 30 minutes (1c, 1d) and 2 hours (1e, 1f) ) This is the result of analyzing the degree of intracellular permeability.

2a to 2d show that BEAS-2B cells or HeLa cells were treated with the peptides of the present invention at various concentrations, and analyzed by fluorescence microscopy after 30 minutes in BEAS-2B cells (2a, 2b), and after 2 hours in HeLa cells. These are the results of analysis with a fluorescence microscope (2c, 2d).

3a and 3b show that BEAS-2B cells or HeLa cells were treated with the peptides of the present invention at various concentrations, and the degree of cytotoxicity was analyzed after 24 hours and 48 hours in BEAS-2B cells (3a, 3b), and HeLa cells This is the result of analyzing the degree of cytotoxicity after 24 hours and 48 hours in (3c, 3d).

Hereinafter, the present invention will be described in detail. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1: Construction of cell penetrating peptides

In order to prepare peptides exhibiting cell permeability, each of the peptides of SEQ ID NOs: 1 to 10 shown in Table 4 was prepared to have a purity of 95% or more. This peptide synthesis was carried out by requesting I-Cure Pepgen Co., Ltd.

sequence number	designation	order
SEQ ID NO: 1	BCP 1	MIIFRASEQLDK
SEQ ID NO: 2	BCP 2	MIIFRVLEQSEK
SEQ ID NO: 3	BCP 3	MIIFRIFLHLQK
SEQ ID NO: 4	BCP 4	MIIFRLFLHLNK
SEQ ID NO: 5	BCP 5	MIIFRLSATVKK
SEQ ID NO: 6	BCP 6	MIIFRSTALVKK
SEQ ID NO: 7	BCP 7	MIIFRLSATAKK
SEQ ID NO: 8	BCP 8	MIIFRFYEDAFK
SEQ ID NO: 9	BCP 9	MIIFRKKEDKDK
SEQ ID NO: 10	BCP 10	MIIFRRLRRHKK
SEQ ID NO: 11	TAT (control)	GRKKRRQRRR

Example 2: Fabrication of lipid nanoparticles (1)

In the present invention, a cationic lipid having Formula 1 or a salt thereof as a lipid nanoparticle to be conjugated with a cell-permeable peptide was prepared as follows.

A lipid stock in 100% ethanol was prepared using the lipid identities and molar ratios described below (approximately 7 mg/mL total lipid content).

PEG-C-DMA:CL1:CHOL:DSPC = 1.5:50.0:38.5:10.0

The mRNA was diluted in acetate pH 5 and nuclease-free water to reach a concentration of 0.366 mg/mL mRNA in 100 mM acetate pH 5. Equal volumes of each solution were blended in a T-connector at 400 mL/min using the direct dilution method described in U.S. Patent No. 9,404,127 and diluted with about 4 volumes of PBS, pH 7.4. The formulation was then placed in a Slide-A-Lyzer dialysis unit (MWCO 10,000) and dialyzed overnight in 10 mM Tris, 500 mM NaCl pH 8 (Tris/NaCl buffer). After dialysis, the formulation was concentrated to about 0.6 mg/mL using a VivaSpin concentrator unit (MWCO 100,000) and then filtered through a 0.2 um syringe filter.

Example 3: Fabrication of lipid nanoparticles (2)

In the present invention, cationic lipids of Formulas 2 to 4 or salt formulations thereof as lipid nanoparticles to be conjugated with cell-permeable peptides were prepared as follows, and PCT Publication Nos. WO 2015/199952, WO 2017/004143 and WO 2017/075531 Lipid nanoparticles, cationic lipids and polymer conjugated lipids (PEG-lipids) were prepared and tested according to the general procedures disclosed in (the entire disclosure of which is incorporated herein by reference).

Lipid nanoparticle (LNP)-formulated mRNA was prepared using ionizable amino lipids (cationic lipids), phospholipids, cholesterol and pegylated lipids. LNPs were prepared as follows. Cationic lipids, DSPC, cholesterol and PEG-lipids were dissolved in ethanol in molar ratios of approximately 50:10:38.5:1.5 or 47.5:10:40.8:1.7. LNPs for the examples included, for example, cationic lipid compounds 2-3 and the aforementioned components. Lipid nanoparticles (LNPs) containing compounds 2-3 were prepared at an mRNA to total lipid ratio of 0.03 to 0.04 w/w. Briefly, mRNA was diluted to 0.05-0.2 mg/mL in 10-50 mM citrate buffer, pH 4. The ethanolic lipid solution was mixed with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) using a syringe pump at a total flow rate exceeding 15 ml/min. Ethanol was then removed, and the external buffer was replaced by PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The particle diameter size of the lipid nanoparticles, determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK), was between 60 and 90 nm. For other cationic lipid components mentioned herein, the method of formulation is similar.

In particular, a cationic lipid of Formula 2-3 or a salt formulation thereof as a lipid nanoparticle to be conjugated with a cell-permeable peptide in the present invention was prepared as follows.

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid (0.33 g) and 4-aminobutan-1-ol (0.23 g) in methylene chloride (20 mL) was added for 2 h. while treated with sodium triacetoxyborohydride (1.3 g). This solution was washed with aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent was removed. The residue was passed through a silica gel column using a methanol/methylene chloride (0 to 8/100 to 92%) gradient to give compound 2-3 as a colorless oil (0.4 g).

Example 4: Fabrication of Lipid Nanoparticle and Cell Penetrating Peptide Complex

The peptide (40 mg, 0.023 mmol) synthesized in Example 1 was dissolved in milliQ water (0.25 mL). (Boc) ₂ O (15 mg, 0.069 mmol) dissolved in 1,4-dioxane was added to this solution along with triethylamine (10 μL). The reaction mixture was stirred at room temperature for 6 hours. After removing the solvent under reduced pressure, the residue was suspended in CHCl ₃ solution and then washed with water. After drying the CHCl ₃ solution with MGSO ₄ , the solvent was removed.

Boc-peptide and dioleoyl phosphatidyl ethanol amine (DOPE) were dissolved in N-methyl pyrrolidone/CH ₂ Cl ₂ . To this solution was added BOP followed by triethylamine (10 μL) and the mixture was stirred for 6 hours. The solvent was removed under reduced pressure at 37°C. After the residue was suspended in MeOH, the solution was filtered. After solvent removal, the residue was resuspended in acetonitrile and centrifuged. The resulting pellet was subjected to column chromatography using a Sep-Pak C18 Cartridge (Waters) to purify Boc-peptide-DOPE. Boc-peptide-DOPE (27.3 mg, 0.011 mmol) was dissolved in TFA (1 mL) and stirred for 1.5 h. TFA was removed under reduced pressure to obtain DOPE-peptide conjugate (25.7 mg).

Protamine sulfate (40 μg) or palmitoyl derivative (22 μg) of the peptide was incubated with siRNA in RNase free water (1 mL) at 25° C. for 20 minutes to obtain a cationic core. Meanwhile, DOPE, cholesterol, and DMPG (dimyristoyl phosphatidyl glycerol) dissolved in chloroform were evaporated under reduced pressure and stored in vacuum for 1 hour. LNP-encapsulated siRNA was prepared by hydrating a thin lipid film with RNase free water (1 mL) containing a cationic core. LNP-siRNA was purified using a Sepharose ^TM 4 Fast Flow column. The absorbance at 260 and 750 nm of each fraction was monitored as an indicator of siRNA and LNP, respectively. Peptide-DOPE dissolved in RNase-free water was added to the LNP-siRNA solution, followed by incubation at 40 °C for 30 min to transform LNP into CPP.

Example 5: Confirmation of intracellular penetration pattern of cell penetrating peptides

Intracellular penetration patterns of the peptides of the present invention were confirmed by fluorescence microscopy.

To this end, rhodamine fluorescent dye was labeled at the amino terminus of each peptide of SEQ ID NOs: 1 to 10 prepared in Example 1. BEAS-2B cells or HeLa cells were treated with peptides of P1 to P10 at various concentrations, and then the concentration of peptides in cells was evaluated after 15 minutes, 30 minutes, and 2 hours. As a control, a TAT peptide (GRKKRRQRRR) (SEQ ID NO: 11) was used.

As shown in Figure 1, it was confirmed that all of the peptides P1 to P10 of the present invention showed significantly improved cell permeability than TAT used as a control.

Then, BEAS-2B cells or HeLa cells were treated with peptides P3, P4, and P5 at concentrations of 10 μM and 50 μM, and control TAT was also treated at the same concentration. However, at the time of cell inoculation, the cell culture slide was inoculated, because plastic causes fluorescence interference when observed under a fluorescence microscope. After washing, the glass cover glass was adhered to the slide glass using mounting medium with DAPI, and the sample was observed under a fluorescence microscope 400 times.

As shown in FIG. 2, it was confirmed that P3, P4, and P5 all showed significantly improved cell permeability than TAT used as a control.

Example 6: Confirmation of cytotoxicity of cell penetrating peptides

In order to confirm whether the intracellular permeation activity of the peptide of the present invention was caused by weakening of the cell membrane due to cytotoxicity, cytotoxicity was confirmed as follows.

Cells were treated with peptides prepared in the same manner as in Example 2 in the culture medium at different concentrations (0, 1, 10, 100 μM), and treated for 24 hours and 48 hours. 10ul of CCK-8 was added to each well and after 3 hours in a 5% CO ₂ incubator maintained at 37°C and humidified conditions, absorbance at 460 nm using a microplate absorbance reader (SYNERGY H1 microplate reader, BIO-TEK) was measured.

As shown in FIG. 3, no cytotoxicity was exhibited up to a concentration of 10 μM, and even at a concentration of 100 μM, cytotoxicity to a degree that could affect the results of the cell permeability test was not confirmed.

The complex of the lipid nanoparticle and the novel cell-penetrating peptide of the present invention can effectively penetrate into living organisms such as cells, tissues, and blood, and thus has very high industrial applicability.

Claims (14)

1. A complex comprising a lipid nanoparticle and a cell penetrating peptide consisting of the following amino acid sequence:

   MIIFR-R1-R2-R3-R4-R5-R6

   In the above formula,

   R1 is any one amino acid selected from the group consisting of A, V, I, L, S, F, K and R,

   R2 is any one amino acid selected from the group consisting of S, L, F, T and Y;

   R3 is any one amino acid selected from the group consisting of E, L, A and R,

   R4 is any one amino acid selected from the group consisting of Q, H, T, L, D and R,

   R5 is any one amino acid selected from the group consisting of L, S, V, A, K and H;

   R6 is any one dipeptide selected from the group consisting of DK, EK, QK, NK, KK and FK.
2. The complex of claim 1, wherein the lipid nanoparticle comprises:

   (i) one or more nucleic acid molecules; (ii) cholesterol; (iii) DSPC; (iv) PEG-C-DNA; and (v) a cationic lipid of Formula 1:

   [Formula 1]

   

   or as a salt thereof,

   where the molar percentages of total lipids relative to PEG-C-DNA, cationic lipids, cholesterol, and DSPC are:

   PEG-C-DNA: about 1.5;

   Cationic lipids: about 50.0;

   Cholesterol: about 38.5; and

   DSPC: around 10.0.
3. The complex of claim 1, wherein the lipid nanoparticle comprises:

   (i) a cationic lipid having Formula 2:

   [Formula 2]

   

   Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

   L ¹ and L ² are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond;

   R ^1a and R ^1b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^1a is H or C ₁ -C ₁₂ alkyl, and R ^1b is the carbon atom to which it is attached. together with adjacent R ^1b and the carbon atom to which it is attached to form a carbon-carbon double bond;

   R ^2a and R ^2b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^2a is H or C ₁ -C ₁₂ alkyl, and R ^2b is the carbon atom to which it is attached. together with adjacent R ^2b and the carbon atom to which it is attached to form a carbon-carbon double bond;

   R ^3a and R ^3b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^3a is H or C ₁ -C ₁₂ alkyl, and R ^3b is the carbon atom to which it is attached. together with adjacent R ^3b and the carbon atom to which it is attached to form a carbon-carbon double bond;

   R ^4a and R ^4b are, at each occurrence, independently (a) H or C ₁ -C ₁₂ alkyl, or (b) R ^4a is H or C ₁ -C ₁₂ alkyl, and R ^4b is the carbon atom to which it is attached. together with adjacent R ^4b and the carbon atom to which it is attached to form a carbon-carbon double bond;

   R ⁵ and R ⁶ are each independently methyl or cycloalkyl;

   R ⁷ , at each occurrence, is independently H or C ₁ -C ₁₂ alkyl;

   R ⁸ and R ⁹ are each independently C ₁ -C ₁₂ alkyl; R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle containing one nitrogen atom;

   a and d are each independently an integer of 0 to 24;

   b and c are each independently an integer from 1 to 24;

   e is 1 or 2); and

   (ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

   A complex comprising a.
4. The complex of claim 1, wherein the lipid nanoparticle comprises:

   (i) a cationic lipid having Formula 3:

   [Formula 3]

   

   Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

   L ¹ and L ² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -SS- , -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, -NR ^a C(=O)NRa-, - OC(=O)NR ^a -, -NR ^a C(=O)O-, or a direct bond;

   G ¹ is C ₁ -C ₂ alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR ^a C(=O)- or a direct bond;

   G ² is -C(=0)-, -(C=0)0-, -C(=0)S-, -C(=0)NR ^a - or a direct key;

   G ³ is C ₁ -C ₆ alkylene;

   R ^a is H or C ₁ -C ₁₂ alkyl;

   R ^1a and R ^1b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^1a is H or C ₁ -C ₁₂ alkyl, R ^1b taken together with the carbon atom to which it is attached, together with adjacent R ^1b and the carbon atom to which it is attached, form a carbon-carbon double bond;

   R ^2a and R ^2b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^2a is H or C ₁ -C ₁₂ alkyl, and R ^2b is taken together with the carbon atom to which it is attached, together with adjacent R ^2b and the carbon atom to which it is attached, to form a carbon-carbon double bond;

   R ^3a and R ^3b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^3a is H or C ₁ -C ₁₂ alkyl, and R ^3b is taken together with the carbon atom to which it is attached, together with adjacent R ^3b and the carbon atom to which it is attached, to form a carbon-carbon double bond;

   R ^4a and R ^4b are, at each occurrence, independently: (a) H or C ₁ -C ₁₂ alkyl; or (b) R ^4a is H or C ₁ -C ₁₂ alkyl, and R ^4b is taken together with the carbon atom to which it is attached, together with adjacent R ^4b and the carbon atom to which it is attached to form a carbon-carbon double bond;

   R ⁵ and R ⁶ are each independently H or methyl;

   R ⁷ is C ₄ -C ₂₀ alkyl;

   R ⁸ and R ⁹ are each independently C ₁ -C ₁₂ alkyl; R ⁸ and R ⁹ together with the nitrogen atom to which they are attached form a 5, 6 or 7 membered heterocycle;

   a, b, c and d are each independently an integer from 1 to 24;

   x is 0, 1 or 2; and

   (ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

   A complex comprising a.
5. The complex of claim 1, wherein the lipid nanoparticle comprises:

   (i) a cationic lipid having Formula 4:

   [Formula 4]

   

   Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (in the formula:

   L ¹ or L ² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -SS- , -C(=O)S-, -SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, -NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O-;

   Preferably L ¹ or L ² is -O(C=O)- or -(C=O)O-;

   G ¹ and G ² are each independently unsubstituted C ₁ -C ₁₂ alkylene or C ₁ -C ₁₂ alkenylene;

   G ³ is C ₁ -C ₂₄ alkylene, C ₁ -C ₂₄ alkenylene, C ₃ -C ₈ cycloalkylene, or C ₃ -C ₈ cycloalkenylene;

   R ^a is H or C ₁ -C ₁₂ alkyl;

   R ¹ and R ² are each independently C ₆ -C ₂₄ alkyl or C ₆ -C ₂₄ alkenyl;

   R ³ is H, OR ⁵ , CN, -C(=0)OR ⁴ , -OC(=0)R ⁴ or -NR ⁵ C(=0)R ⁴ ;

   R ⁴ is C ₁ -C ₁₂ alkyl;

   R ⁵ is H or C ₁ -C ₆ alkyl;

   x is 0, 1 or 2; and

   (ii) an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, wherein the mRNA compound is optionally free of nucleoside modifications, in particular free of base modifications; The mRNA compound is an mRNA compound that is encapsulated in the lipid nanoparticle or associated with the lipid nanoparticle;

   A complex comprising a.
6. The complex according to claim 1, wherein the peptide is selected from the group consisting of SEQ ID NOs: 1 to 10.
7. A composition for promoting cell permeation of a substance comprising the complex of any one of claims 1 to 6.
8. The method of claim 7, wherein the cells are epithelial cells, muscle cells, nerve cells, glandular cells, glial cells, germ cells, stem cells, mesenchymal cells, mesenchymal stem cells, osteoblasts, osteocytes, osteoblasts, osteoclasts, A composition characterized in that it is selected from the group consisting of blood cells, hematopoietic cells, lung cells, hepatocytes, fibroblasts, immune cells, endothelial cells, adipocytes, and chondrocytes.
9. 8. The composition according to claim 7, wherein the composition is for delivering a biologically active substance through mucous membranes or skin.
10. Use of a material comprising the complex of any one of claims 1 to 6 for preparing a composition for promoting cell permeation.
11. The method of claim 10, wherein the cells are epithelial cells, muscle cells, nerve cells, glandular cells, glial cells, germ cells, stem cells, mesenchymal cells, mesenchymal stem cells, osteoblasts, osteocytes, osteoblasts, osteoclasts, Use characterized by being selected from the group consisting of blood cells, hematopoietic cells, lung cells, hepatocytes, fibroblasts, immune cells, endothelial cells, adipocytes, and chondrocytes.
12. 11. The use according to claim 10, wherein the composition is for delivery of a biologically active substance through mucous membranes or skin.
13. A method for promoting cell permeation comprising administering an effective amount of a composition comprising a substance comprising the complex of any one of claims 1 to 6 as an active ingredient to a subject in need thereof.
14. The method of claim 13, wherein the cells are epithelial cells, muscle cells, nerve cells, glandular cells, glial cells, germ cells, stem cells, mesenchymal cells, mesenchymal stem cells, osteoblasts, osteocytes, osteoblasts, osteoclasts, A method characterized by being selected from the group consisting of blood cells, hematopoietic cells, lung cells, hepatocytes, fibroblasts, immune cells, endothelial cells, adipocytes, and chondrocytes.

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