Synthesis of peptide-based ionizable lipid and use thereof

Abstract

The present invention relates to the synthesis of a peptide-based ionizable lipid and the use thereof, and specifically relates to an amino acid molecular building block with an alkylated side-chain primary amino group, a peptide-based ionizable lipid (PIL) formed therefrom, a method for synthesizing PIL, and a PIL library containing a plurality of PILs. The present invention further relates to a lipid nanoparticle (LNP) containing PIL, in particular a PIL-mediated organ-targeted LNP (PILOT LNP), a pharmaceutical composition containing same, and a targeted delivery method for administering the LNP or the pharmaceutical composition, or a method for preventing or treating a disease.

Description

Synthesis and applications of peptidyl ionizable lipids TECHNICAL FIELD

[0001] The present invention relates to ionizable lipids, lipid nanoparticles, and methods of making and using the same, and in particular to side chain primary aminoalkylated amino acid building blocks and peptidyl ionizable lipids formed therefrom. BACKGROUND

[0002] Messenger ribonucleic acid (mRNA) is a single-stranded ribonucleic acid that can carry genetic information and guide protein synthesis, which is transcribed from deoxyribonucleic acid (DNA) as a template. By encoding specific pathogen antigens, cancer antigens, gene editing components, or other disease-related therapeutic proteins, mRNA can be used in various fields, including vaccine development, gene editing, and protein replacement therapy. In addition, with the rapid development of mRNA engineering technologies such as chemical modification, sequence optimization, production purification, the limitations of the initial mRNA, such as instability, high immunogenicity, and expensive production cost, have been gradually solved, which also lays the foundation for mRNA as a drug and vaccine. However, in clinical transformation, mRNA needs to be expressed in cells in vivo, but it is a huge, negatively charged nucleic acid molecule, which is difficult to enter cells through the biological membrane of phospholipid bilayer with the same negative charge. At the same time, the abundant nucleases in blood and tissue fluid will rapidly degrade the mRNA molecule, making it lose its due function. Therefore, the development of efficient mRNA delivery vectors is the key and difficulty of mRNA drug research and development.

[0003] An ideal mRNA carrier needs to protect mRNA from degradation by nucleases, while enabling efficient delivery of mRNA to the cytoplasm across various biological barriers, and meeting the storage conditions and time limit of mRNA drugs. Lipid nanoparticles (LNP) is the most advanced mRNA delivery carrier in clinical research, which has significant advantages in mRNA encapsulation efficiency, in vivo transfection ability, and carrier stability. Currently, two mRNA vaccines for COVID-19 and more than 90% of mRNA drugs in clinical research use LNP delivery technology. The classic LNP carrier is usually composed of ionizable lipids, cholesterol, auxiliary phospholipids, and polyethylene glycol lipids. It can be efficiently taken up by antigen-presenting cells and express the encoded antigen after local muscle injection, while it is targeted to the liver site through adsorption of apolipoprotein E (ApoE) in the blood after intravenous injection, and enters the liver parenchymal cells through low-density lipoprotein (LDLR)-mediated endocytosis. The liver-specific delivery property of LNP carrier has great advantages for the development of mRNA therapy for liver-related diseases, and current clinical researches are mainly focused on liver diseases. However, at the same time, it also limits the application of LNP delivery technology in the treatment of diseases in organs outside the liver. In order to fully exert the therapeutic potential of mRNA drugs, it is urgent to develop LNP carriers that can specifically deliver mRNA to organs outside the liver.

[0004] Currently, there are mainly three methods to achieve LNP delivery to extrahepatic organs: 1) modifying ligands (including antibodies, polypeptides, aptamers, small molecule ligands, etc.) with organ or cell targeting ability on the surface of LNP; 2) adding additional lipid components such as permanent cationic lipids or anionic lipids to the classic 4-component LNP; 3) developing ionizable lipids with extrahepatic organ targeting ability. Each of the above strategies has its own advantages and disadvantages. The ligand modification strategy has good specificity, but involves a series of steps such as ligand screening, ligand connection condition optimization, and LNP purification after modification, which is complicated and costly. The additional lipid strategy is simple and universal, but the additional components (such as cationic lipids (2,3-dioleoyl-propyl)-trimethylamine, DOTAP) may increase the toxicity of LNP carriers, making LNP prescription optimization more complex. Compared with the first two, the development of extrahepatic targeting ionizable lipids can to some extent avoid the above disadvantages, so it has greater application potential. However, the current development of extrahepatic targeting ionizable lipids lacks corresponding theoretical guidance and still relies heavily on high-throughput screening of lipid libraries. The current common lipid molecular structure is relatively similar, and the amine core they contain is mostly tertiary amine, and the hydrophobic tail is different number and type of fatty chains. This similarity in structure results in most of the final lipid molecules still being liver-targeted, with only a few lipids having extrahepatic organ targeting ability. Therefore, it is of great significance to construct a library of ionizable lipids with more diverse chemical structures, more easily controllable physicochemical properties, and simpler synthesis methods for organ-targeted mRNA delivery.

[0005] Polypeptides are a class of compounds composed of amino acids connected by peptide bonds. Due to the structural diversity, controllable sequence length, high combinability, and diverse chemical and biological functions of natural and synthetic amino acid polypeptides, they have a wide range of applications in targeted peptides, cell-penetrating peptides, vaccine antigen peptides, agonists, antagonists, and cytotoxic agents. Inspired by this and based on the research background in the above fields, the inventors propose a new design of ionizable cationic lipids in this application: based on different types and numbers of amino acid building blocks, a library of peptide-based ionizable lipids (PIL) with diverse structures and adjustable properties is constructed by introducing diverse hydrophobic carbon chains, and finally LNP based on PIL is used for organ-specific mRNA delivery and therapy.

[0006] SUMMARY

[0007] In a first aspect, the present application provides an amino acid molecular building block having the following formula I structure

[0008] wherein,

[0009] m is an integer of 0-10, preferably 1-4,

[0010] A1and A2are hydrophobic tails and are independently of each other an optionally substituted, saturated or unsaturated, linear or branched alkyl chain having a carbon number of 4 to 25, wherein the alkyl chain optionally contains one or more linking groups L selected from an amide bond, an ester bond, a disulfide bond, a kethlmercapto bond, an ether bond, or a combination thereof,

[0011] A3is an amino protecting group, and

[0012] A4is a hydrogen atom or an alkyl group.

[0013] In the present application, m is an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, m is 1, i.e., the amino acid building block of the present application is an amino acid building block in which the side chain primary amino group of 2,3-diaminopropionic acid (Dap) is alkylated. In certain embodiments, m is 2, i.e., the amino acid building block of the present application is an amino acid building block in which the side chain primary amino group of 2,4-diaminobutyric acid (Dab) is alkylated. In certain embodiments, m is 3, i.e., the amino acid building block of the present application is an amino acid building block in which the side chain primary amino group of ornithine is alkylated. In certain embodiments, m is 4, i.e., the amino acid building block of the present application is an amino acid building block in which the side chain primary amino group of lysine is alkylated. In certain embodiments, m is preferably 1, 2, 3, or 4, more preferably 2, 3, or 4.

[0014] In certain embodiments, A1or A2has a carbon number of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In certain embodiments, when A1and A2contain one or more linking groups L, the carbon number of the alkyl chain refers to the number of carbon atoms contained in the hydrocarbon group attached after the last linking group. In certain embodiments, when A1and A2are an amide bond-containing alkyl chain or an ester bond-containing alkyl chain, the carbon number refers to the number of carbon atoms contained in the hydrocarbon group attached after the amide bond or the ester bond.

[0015] In certain preferred embodiments, the alkyl chain of A1or A2of the present application contains 0, 1, 2, 3, 4, 5, or 6 linking groups L. In certain preferred embodiments, the linking group L can be a biodegradable group, such as an ester bond that is stable at physiological pH but is hydrolyzed by enzymes within tissues and cells, a disulfide bond that is sensitive to reduced intracellular environment, a kethlmercapto bond that is responsive to ROS, etc.

[0016] In certain embodiments, the amino protecting group of the present application is fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), p-methoxybenzyl (PMB), benzyl (Bn), trityl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), N-l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), or allyloxycarbonyl (Alloc).

[0017] In certain embodiments, A4is a hydrogen atom or an alkyl group. Preferably, A4is a hydrogen atom or a lower alkyl group, such as methyl, ethyl, or propyl.

[0018] In certain embodiments, Ai and A2are independently of each other -(B1-L) s -B2, wherein s is an integer from 0 to 6, such as 0, 1, 2, 3, 4, 5, or 6, wherein B1is independently in each (B1-L) unit either absent or an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25, L is independently in each (B1-L) unit either absent or a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethal bond, an ether bond, or a combination thereof, and B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25.

[0019] In certain embodiments, B1is absent in the (B1-L) unit, i.e., the (B1-L) unit is -L-. In certain embodiments, B1is independently at each occurrence an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25, preferably B1is an optionally substituted linear or branched alkyl, alkenyl, or alkynyl group having a carbon number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In certain embodiments, B1is hydroxyl-substituted.

[0020] In certain embodiments, L is absent in the (B1-L) unit, i.e., the (B1-L) unit is B1. In certain embodiments, L is independently at each occurrence a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethal bond, an ether bond, or a combination thereof.

[0021] In certain embodiments, B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25, preferably B2is an optionally substituted linear or branched alkyl, alkenyl, or alkynyl group having a carbon number from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In certain embodiments, B2is hydroxyl-substituted.

[0022] In certain embodiments, A1and A2are independently of each other -B1-L-B2, -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, -B1-COO-B1-L-B2, or -B1-OOC-B1-L-B2, wherein each B1is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25, wherein L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof, B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25.

[0023] In certain embodiments, A1and A2are independently of each other selected from: a saturated alkyl chain, an unsaturated alkyl chain, a hydroxyl-containing alkyl chain, an amide bond-containing alkyl chain, an ester bond-containing alkyl chain, a disulfide bond-containing alkyl chain, a kethol thioacetal-containing alkyl chain, a branched alkyl chain.

[0024] In certain embodiments, A1and A2are independently of each other selected from: -CH2CH2(CH2) q CH2CH3, -CH2CHOH(CH2) q CH2CH3, -CH2CH2CONHCH2CH2(CH2) q CH2CH3, -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, -CH2CH2COOCH2CH2(CH2) q CH2CH3, -CH2CH2OOCCH2CH2(CH2) q CH2CH3, -CH2CH2OOCCH2CH2(CH2) wherein q is an integer from 0 to 21, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21, preferably q is an integer from 4 to 10.

[0025] In certain embodiments, A1and A2are the same.

[0026] In a second aspect, the present application provides a peptide-based ionizable lipid (PIL) having the following structure II,

[0027] wherein,

[0028] A4is as defined in the first aspect,

[0029] n is an integer from 1 to 30,

[0030] X is O or S,

[0031] R1represents the N-terminus of the PIL, and R1is a hydrogen atom or has an acetyl, an amino acid, and / or other functional group modification,

[0032] R2represents a pendant group contained by the PIL, R2in each occurrence is independently a pendant group of an amino acid building block according to any one of claims 1-5 a natural or unnatural amino acid pendant group, and at least one R2in the PIL is

[0033] R3represents the C-terminus of the PIL, and R3is a hydroxyl group or has an amino, amino acid and / or other functional group modification.

[0034] In certain embodiments, R2represents a pendant group contained by the PIL, in each unit, R2is independently selected from the pendant groups of the amino acid building blocks of the first aspect a natural or unnatural amino acid pendant group.

[0035] In certain embodiments, at least 1, 2, 3, 4 or 5 R2in the PIL of the present application is a pendant group of the amino acid building blocks of the first aspect

[0036] In certain embodiments, a natural amino acid pendant group is, for example, a hydrogen atom, a methyl group, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH2SCH3, -CH2C6H5, -CH2C6H4OH, -CH2C8H6N, -CH2OH, -CH(OH)CH3, -CH2SH, -CH2COOH, -CH2CH2COOH, -CH2CONH2, -CH2CH2CONH2, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2C3H3N2, or -CH2CH2CH2- (forming a ring structure with the amino group).

[0037] In certain embodiments, the non-natural amino acid side group refers to other non-natural amino acid side groups than the amino acid building block side groups formed by the primary aminoalkylation defined in the first aspect of the application, such as for example the amino acid side groups of N-ethyl beta-alanine (Neb), sarcosine (Sar), alpha, beta-diaminopropionic acid (Adp), beta-amino-N-butyric acid (aBut or Bab), beta-aminoisobutyric acid (Bai), alpha-aminoisobutyric acid (Aib), gamma-aminobutyric acid (Gab), alpha-aminobutyric acid (Anb), N-methylalanine (Nma), N-ethylglycine (Neg), allothreonine (Alt), Hse, 4-amino-3-hydroxybutyric acid (Hga), 2,4-diaminobutyric acid (Dab), hydroxyproline (Hyp), isovaline (Iva), norvaline (Nor), L-cyclopropylglycine (Cpg), N-propylglycine (Npg), homocysteine (Hcy), piperidinic acid (Pip), ornithine (Orn), tert-leucine (Tle), alloisoleucine (Ali), norleucine (Nle), 2-aminoheptanoic acid (Ahe) or citrulline (Cit).

[0038] In certain embodiments, n is an integer from 1 to 30, such as n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably n is 1, 2, 3, 4 or 5.

[0039] In certain embodiments, A1 and A2 are the same in the same amino acid building block monomer. In certain embodiments, both A1 and A2 are hydroxyl-containing alkyl chain containing amino acid building block monomers in an amount of less than or equal to 2.

[0040] In certain embodiments, A1 and A2 are alkyl chains having a carbon number of 8-14 and n is 3-5, preferably A1 and A2 are alkyl chains having a carbon number of 12 and n is 3 or 4, further preferably n is 3-5, m is 2-4 and A1 and A2 are saturated alkyl chains having a carbon number of 10-14, more preferably n is 4, m is 2 and A1 and A2 are saturated alkyl chains having a carbon number of 12.

[0041] In certain embodiments, in the PIL of the application:

[0042] (i) n is 4-5,

[0043] (ii) n is 3 and A1 and A2 are alkyl chains having a carbon number of 10-14,

[0044] (iii) n is 2 and A1 and A2 are alkyl chains having a carbon number of 16-18,

[0045] (iv) A1and A2are saturated alkyl chains of 12 carbon atoms or hydroxyl-containing alkyl chains, and n is 3-5,

[0046] (v) wherein A1and A2are amide bond-containing alkyl chains of 12 carbon atoms, and n is 3-5, or

[0047] (vi) A1and A2are ester bond-containing alkyl chains of 12 carbon atoms.

[0048] In certain embodiments, the PIL has a free amino group at the N-terminus (R1represents -H) or has an acetylated modification (R1represents -COCH3). In certain embodiments, the PIL has both an acetylated modification and an amino acid modification at the N-terminus (R1represents -(aa)x-COCH3) or has an amino acid modification and a free amino group (R1represents -(aa)x), wherein (aa)x is one or more amino acid residues. In certain embodiments, the PIL has a free carboxyl group at the C-terminus (R3represents -OH) or has an amidated modification (R3represents -NH2). In certain embodiments, the PIL has both an amidated modification and an amino acid modification at the C-terminus (R2represents -(aa)x-NH2) or has an amino acid modification and a free carboxyl group (R2represents -(aa)x), wherein (aa)x is one or more amino acid residues.

[0049] In certain embodiments, the amino acid that modifies the N-terminus and / or the C-terminus of the PIL is a natural amino acid or a non-natural amino acid. In certain embodiments, the amino acid that modifies the N-terminus and / or the C-terminus of the PIL is a natural amino acid, including but not limited to alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V).

[0050] In certain embodiments, the PIL has 1 to 10 amino acid modifications at the N-terminus and / or the C-terminus, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0051] In certain embodiments, the other functional modification is any organ targeting small molecule. In certain embodiments, the other functional group modification refers to a phosphorylation modification, preferably a bisphosphonate modification, more preferably an alendronate modification, for example malonylated alendronate modification, for example

[0052] In certain embodiments,

[0053] (a) the PIL has a basic amino acid modification, preferably a basic amino acid modification at the N-terminus and an acetylation modification;

[0054] (b) the PIL has a basic amino acid modification at the N-terminus and a free amino group;

[0055] (c) the PIL has a proline modification, preferably a proline modification at the N-terminus and a free amino group;

[0056] (d) the PIL has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine or a combination thereof, preferably an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine or a combination thereof at the N-terminus and an acetylation modification;

[0057] (e) the PIL has a tryptophan modification, preferably a tryptophan modification at the N-terminus and an acetylation modification;

[0058] (f) the PIL has an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine or a combination thereof, preferably an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine or a combination thereof at the N-terminus and an acetylation modification;

[0059] (g) the PIL has an acidic amino acid modification, preferably an acidic amino acid modification at the C-terminus and a free carboxyl group; or

[0060] (h) the PIL has a phosphorylation modification, for example a phosphorylation modification at the N-terminus or C-terminus, preferably a bisphosphonate modification, more preferably an alendronate modification.

[0061] In certain embodiments, the PIL of the application is selected from:

[0062] In a third aspect, the present application provides a method of synthesizing a PIL, the method comprising:

[0063] (A) coupling a solid-phase synthesis support to an amino acid building block and removing an amino protecting group, and

[0064] (B) adding an amino acid building block to a free amino group and removing an amino protecting group, repeating this step to extend the PIL chain, wherein one or more of the amino acid building blocks in step (A) or (B) is the amino acid building block of the first aspect,

[0065] (C) optionally performing acetylation, and

[0066] (D) cleaving the synthesized PIL chain from the solid-phase synthesis support.

[0067] In certain embodiments, the solid-phase synthesis support is a resin, such as Rink Amide resin or 2-chlorotrityl chloride (2-CTC) resin.

[0068] In a fourth aspect, the present application provides a library of PILs comprising a plurality of the PILs of the second aspect.

[0069] In a fifth aspect, the present application provides a lipid nanoparticle (LNP) comprising the peptide-based ionizable lipid of the second aspect, optionally an auxiliary lipid, and optionally an active agent.

[0070] In certain embodiments, the helper lipid comprises a phospholipid, a sterol, and / or a PEG lipid.

[0071] In certain embodiments, the lipid nanoparticle comprises, based on total lipids, about 10 mol% to about 100 mol% of a peptide-based ionizable lipid, about 0 mol% to about 30 mol% of a phospholipid, about 0 mol% to about 70 mol% of a sterol, and / or about 0 mol% to about 10 mol% of a PEG lipid.

[0072] In certain embodiments, the LNP is an organ-targeting LNP, wherein the target organ is selected from the group consisting of: lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin, preferably the LNP is a spleen-targeting LNP, a liver-targeting LNP, a lung-targeting LNP, or a bone-targeting LNP.

[0073] In certain embodiments, the LNP is a spleen-targeting LNP, and wherein the LNP has one or more of the following characteristics:

[0074] (i) in the PIL comprised by the LNP, n is 2 and A1and A2are alkyl chains having a carbon atom number of 16-18;

[0075] (ii) in the PIL comprised by the LNP, A1and A2are amide bond-containing alkyl chains having a carbon atom number of 12, and n is 3-5;

[0076] (iii) the PIL comprised by the LNP has a single free amino group or a free carboxyl group at the terminus;

[0077] (iv) the PIL comprised by the LNP has a basic amino acid modification, preferably the PIL has a basic amino acid modification and an acetylation modification at the N-terminus;

[0078] (v) the PIL comprised by the LNP has a proline modification, preferably the PIL has a proline modification and a free amino group at the N-terminus;

[0079] (vi) the PIL comprised by the LNP has a tryptophan modification, preferably the PIL has a tryptophan modification and an acetylation modification at the N-terminus; or

[0080] (vii) the PIL comprised by the LNP has an acidic amino acid modification, preferably the PIL has an acidic amino acid modification and a free carboxyl group at the C-terminus, preferably wherein the number of acidic amino acid modifications is less than or equal to 5, less than or equal to 4, or less than or equal to 3.

[0081] In certain embodiments, the LNP is a liver-targeting LNP, and wherein the LNP has one or more of the following characteristics:

[0082] (i) in the PIL comprised by the LNP, n is 4-5, or n is 3 and A1and A2are alkyl chains having a carbon atom number of 10-14;

[0083] (ii) in the PIL comprised by the LNP, A1and A2are saturated alkyl chains or hydroxyl-containing alkyl chains having a carbon atom number of 12, and n is 3-5; or

[0084] (iii) the PIL comprised by the LNP has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof, preferably the PIL has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof at the N-terminus and an acetylation modification.

[0085] In certain embodiments, the LNP is a lung-targeting LNP, and wherein the PIL comprised by the LNP has a basic amino acid modification, preferably the PIL has a basic amino acid modification at the N-terminus and a free amino group.

[0086] In certain embodiments, the LNP is a liver- and spleen-dual targeting LNP, wherein in the PIL comprised by the LNP, A1and A2are ester bond-containing alkyl chains having a carbon atom number of 12.

[0087] In certain embodiments, the LNP is a bone-targeting LNP, and wherein the PIL comprised by the LNP has a phosphorylation modification, for example the PIL has a phosphorylation modification at the N-terminus or C-terminus, preferably a bisphosphonate modification, more preferably an alendronic acid modification, for example a succinylated alendronic acid modification, for example

[0088] In certain embodiments, the PIL comprised by the LNP has an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine, or a combination thereof at the N-terminus and an acetylation modification.

[0089] In certain embodiments, the active agent is a chemically modified or unmodified nucleic acid molecule, for example a DNA molecule or an RNA molecule, preferably the mass ratio of total lipids to active agent in the LNP is about 1-100: 1.

[0090] In certain embodiments, the active agent of the present application is a chemically modified or unmodified DNA molecule, which can be any type of DNA molecule (but not limited thereto), including but not limited to linear or circular DNA, double-stranded or single-stranded or multi-stranded assembled DNA, coding or non-coding DNA, which is optionally selected from plasmid, oligodeoxynucleotide, genomic DNA, DNA probe, DNA aptamer, DNA nanoframe, DNA primer, homologous repair DNA template, immunostimulatory DNA, or a combination thereof.

[0091] In certain embodiments, the active agent of the present application is a chemically modified or unmodified RNA molecule, which can be any type of RNA molecule (but not limited thereto), including but not limited to messenger RNA (mRNA), small interfering RNA (siRNA), circular RNA (circRNA or oRNA), guide RNA (sgRNA), nicking guide RNA (nicking sgRNA), small hairpin RNA (shRNA), viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), microRNA (miRNA), small nuclear RNA (snRNA), RNA aptamer, antisense RNA, RNA nanoframe, RNA ribonucleoprotein complex, or a combination thereof. In certain embodiments, the active agent of the present application is a chemically modified messenger RNA (mRNA), preferably any mRNA encoding a gene editing element. In preferred embodiments, the active agent of the present application is an mRNA encoding a CRISPR-based gene editing element, such as a prime editing element.

[0092] In certain embodiments, the active agent of the present application can be a combination of the above-mentioned DNA molecules and RNA molecules. In certain embodiments, the active agent is a gene therapy agent. In certain embodiments, the mass ratio of total lipids to active agent in the LNP is about 1-100: 1.

[0093] In a sixth aspect, the present application provides a pharmaceutical composition comprising the LNP according to the fifth aspect and a pharmaceutically acceptable carrier.

[0094] In a seventh aspect, the present application provides a method of delivering an active agent to a target organ or target cell or preventing or treating a disease, the method comprising administering an effective amount of the LNP according to the fifth aspect or the pharmaceutical composition according to the sixth aspect to a subject in need thereof or an ex vivo cell or tissue.

[0095] The present application also provides use of the LNP according to the fifth aspect or the pharmaceutical composition according to the sixth aspect in the manufacture of a medicament for treating or preventing a disease.

[0096] The present application also provides a medicament for treating or preventing a disease, the medicament comprising the LNP of the fifth aspect or the pharmaceutical composition of the sixth aspect.

[0097] The present application also provides the LNP of the fifth aspect or the pharmaceutical composition of the sixth aspect for use in treating or preventing a disease.

[0098] In certain embodiments, the disease is any disease for which the active agent of the present application can have a therapeutic or prophylactic effect. In certain embodiments, the disease is a genetic disease and the active agent is a gene therapy agent. In certain embodiments, the gene therapy agent encodes a CRISPR-based gene editing element such as a prime editing element or a chimeric antigen receptor T immunotherapy agent. In certain embodiments, the active agent is an mRNA capable of eliciting an immune response. In certain embodiments, the disease is a disease requiring organ-targeted therapy. In certain embodiments, the disease is a cancer.

[0099] The present application synthesizes a series of N alpha-fluorenylmethoxycarbonyl (Fmoc)-protected side chain alkylated primary amino alkylated amino acid building blocks (Alkylated Ionizable Fmoc-protected Amino acid, AIFA).

[0100] Secondly, by regulating the number and type of alkylated amino acid building blocks, the present application constructs a library of structurally diverse peptide-based ionizable lipids (PIL) using polypeptide solid-phase synthesis technology.

[0101] Thirdly, in the experiment of delivering mRNA using PIL LNP, the present application not only obtains PILs with high liver delivery efficiency and high specificity, but also finds a general amino acid combination method for regulating organ-specific mRNA delivery of PIL, as follows in detail:

[0102] Feature one, in acetylated PILs at the N-terminus, modification of basic amino acids (such as lysine or arginine) can achieve spleen-targeted mRNA delivery, while modification of basic amino acids in PILs with free amino groups at the N-terminus can achieve lung-targeted mRNA delivery;

[0103] Feature two, in non-acetylated PILs at the N-terminus, proline modification can increase the spleen-specific delivery of mRNA-LNP;

[0104] Feature three, in acetylated PILs at the N-terminus, modification of cysteine, histidine, tyrosine and phenylalanine can enhance the liver-specific delivery of mRNA-LNP; modification of tryptophan can increase the spleen-specific delivery of mRNA-LNP; while modification of other non-charged amino acids can affect its delivery efficiency, but has little effect on its targeting;

[0105] Feature four, in PILs with free carboxyl groups at the carboxyl terminal (C-terminal), modification of acidic amino acids (such as glutamic acid and aspartic acid) can enhance the spleen-specific delivery of mRNA-LNPs.

[0106] Based on the above features, the present application refers to such LNP mediated by PIL for in vivo targeting of organs without additional ligand or lipid molecules as PILOT LNP (Peptide Ionizable Lipid-driven Organ Targeting lipid nanoparticle, FIG. 1). With PILOT LNP, the present application realizes precise delivery of mRNA to extrahepatic organs and mRNA-mediated prime editing (PE).

[0107] In summary, this study synthesized a series of new PILs with novel structures, developed a method for constructing a lipid library with rich structural diversity, and proposed a general amino acid modification strategy to regulate the organ-specific mRNA delivery of PILOT LNP, providing a certain theoretical basis and guidance for the design and synthesis of the next generation of extrahepatic organ-specific ionizable lipids.

[0108] Developing mRNA-LNP delivery vectors targeting extrahepatic organs is one of the key and difficult points in the field of mRNA drug research and development. Current strategies to achieve LNP targeting of extrahepatic organs mainly include modification of ligands, addition of extra lipid components, and development of ionizable lipids with extrahepatic organ targeting ability. Among them, the use of ionizable lipids to achieve extrahepatic targeting has the advantages of simple preparation and optimization process, high reproducibility, relatively low cost, and easy scaling. However, the current screening of extrahepatic targeting ionizable lipids lacks relevant theoretical guidance and design strategies, and its organ specificity is poor and the screening efficiency is low.

[0109] In view of the current situation, the present project proposes to use ionizable lipids as a new type of amino acid building block, to construct a library of peptide-based ionizable lipids (PIL) with rich structures and adjustable properties through the combination of itself and different types of natural amino acids based on solid-phase synthesis technology, and to develop organ-specific mRNA-LNP carriers. Through the synthesis of ionizable alkylated amino acid monomer building blocks (AIFA), the construction of the PIL lipid library, the analysis and establishment of the structure-activity relationship in vitro and in vivo, the present application obtains PIL lipids and corresponding PILOT LNP that can precisely target liver, spleen, lung, bone and other organs. More importantly, the present application discovers a general amino acid combination method for regulating the targeting of PIL lipids to different organs. This amino acid modification strategy has certain guiding significance for the design of a new generation of ionizable lipids with mRNA organ-selective delivery capability. The PIL lipid solid-phase synthesis platform is expected to further accelerate the synthesis and screening of organ-selective ionizable lipids guided by this strategy. In addition, the present application explores and explains the mechanism of the difference in transfection efficiency of PILOT LNP in vitro and in vivo, providing a reference value for the LNP in vitro and in vivo screening strategy in the field. Finally, the present application realizes gene editing and repair by delivering mRNA through PILOT LNP, providing a reliable technology platform for the treatment of genetic diseases.

[0110] The beneficial effects of the present application are summarized as follows:

[0111] (i) A series of Nα-Fmoc group protected primary amino alkylated amino acid building blocks (AIFA) are designed and synthesized, which can be used for solid-phase synthesis of polypeptides based on Fmoc strategy, and help to customize the synthesis of amino acid chains;

[0112] (ii) A series of peptide-based ionizable lipids (PIL) with new structures are obtained by solid-phase synthesis technology: a number of artificially synthesized alkylated amino acid molecular building blocks and natural amino acid molecular building blocks are connected by amide bonds;

[0113] (iii) By regulating the number and type of amino acid building blocks, liver, spleen, lung, and bone-specific targeting PIL structures are obtained, and PILOT LNP is also obtained;

[0114] (iv) A general modification strategy for PIL targeting liver, spleen, lung, and bone is summarized, including: 1) basic amino acid modification can mediate LNP specific targeting of lung or spleen; 2) acidic amino acid modification can mediate LNP specific targeting of spleen; 3) cysteine, histidine, tyrosine and phenylalanine modification can enhance the liver specificity and delivery efficiency of LNP; 4) proline and tryptophan modification can mediate LNP specific targeting of spleen; 5) phosphorylation modification can mediate LNP specific targeting of bone; 6) other non-charge amino acids regulate the delivery efficiency of LNP, etc.

[0115] (v) First use of PILOT LNP to achieve organ-selective PE editing by delivering PE mRNA elements. BRIEF DESCRIPTION OF DRAWINGS

[0116] Figure 1. Schematic of PIL and PILOT LNP.

[0117] Figure 2. Nα-Fmoc protected side chain primary amine alkylated amino acid building blocks.

[0118] Figure 3. Synthetic route of Nα-Fmoc protected alkylated amino acids.

[0119] Figure 4. Representative chemical structures and proton NMR spectra of Nα-Fmoc protected alkylated amino acids.

[0120] Figure 5. Solid phase synthesis of PIL lipids.

[0121] Figure 6. PILs containing 1-3 alkylated (aldehyde reductive amination) lysines.

[0122] Figure 7. PILs containing 4-5 alkylated (aldehyde reductive amination) lysines.

[0123] Figure 8. PILs containing 1-5 alkylated (epoxide ring opening) lysines.

[0124] Figure 9. PILs containing 1-5 alkylated (aldehyde reductive amination) ornithines, 2,4-diaminobutyric acids, 2,3-diaminopropionic acids.

[0125] Figure 10. PILs containing 1-5 alkylated (acrylamide or acrylic ester Michael addition) lysines.

[0126] Figure 11. PILs containing 4 alkylated (aldehyde reductive amination and epoxide ring opening) lysines.

[0127] Figure 12. Solid phase synthetic route of peptidyl ionizable lipid a12K4.

[0128] Figure 13. Structure and proton NMR spectrum of peptidyl ionizable lipid a12K4.

[0129] Figure 14. Lipid composition of mRNA-LNP formulations.

[0130] Figure 15. Microfluidic preparation of PIL-based mRNA-LNPs.

[0131] Figure 16. Encapsulation efficiency of Luc mRNA by PIL LNP.

[0132] Figure 17. Hydrated particle size, surface charge and polydispersity index of LNP prepared with a6K1-a18K5 PIL lipids.

[0133] Figure 18. Hydrated particle size, surface charge and polydispersity index of LNP prepared with PIL lipids containing different number of a and e alkyl chains.

[0134] Figure 19. Hydrated particle size, surface charge and polydispersity index of LNP prepared with PIL lipids containing different a12alkyl chain amino acids.

[0135] Figure 20. Plot of TNS fluorescence value versus pH value in LNP solution at different pH.

[0136] Figure 21. Apparent pKa value of LNP prepared with a6K1-a18K5 PIL lipids.

[0137] Figure 22. Cryo-EM image of a12K4 LNP.

[0138] Figure 23. Structure-activity relationship of PILs.

[0139] Figure 24. General structure of PILs containing 1-5 a or e alkylated lysines.

[0140] Figure 25. Bioluminescence imaging of whole body and organs of mice mediated by Luc mRNA-LNP.

[0141] Figure 26. Heat map of liver site fluorescence intensity mediated by Luc mRNA-LNP.

[0142] Figure 27. Comparison of liver site fluorescence intensity mediated by PILs with different alkyl chain length.

[0143] Figure 28. Comparison of liver site fluorescence intensity mediated by PILs containing different number of AIFA.

[0144] Figure 29. Comparison of liver site fluorescence intensity mediated by PILs with different alkyl type, same alkyl chain length and number of AIFA.

[0145] Figure 30. Comparison of liver site fluorescence intensity mediated by PILs containing different number of a and e alkyl chains.

[0146] Figure 31. Lysines and their analogues with different side chain length and side chain amino pKa value.

[0147] Figure 32. Comparison of liver site fluorescence intensity mediated by PILs containing different lysine analogues.

[0148] Figure 33. Relationship between PIL structure and organ-selective delivery of mRNA-LNP.

[0149] Figure 34. Heatmap of the ratio of fluorescence intensity of liver and spleen to the total fluorescence intensity of liver and spleen for different PILs.

[0150] Figure 35. PILs with different alkyl chain types.

[0151] Figure 36. The percentage of mRNA expression in liver, spleen, and lung mediated by PILs with different alkyl chain types.

[0152] Figure 37. Schematic diagram of PILs with different end groups prepared using different resins and synthesis methods.

[0153] Figure 38. a12K2 and a12K4 lipids with different end groups.

[0154] Figure 39. The percentage of mRNA expression in liver, spleen, and lung mediated by PILs with different end groups.

[0155] Figure 40. Chemical structure of a12K2 modified with different natural amino acids.

[0156] Figure 41. The percentage of mRNA expression in liver, spleen, and lung mediated by a12K2 modified with basic and acidic amino acids.

[0157] Figure 42. The percentage of mRNA expression in liver, spleen, and lung mediated by a12K2 modified with special and aromatic amino acids.

[0158] Figure 43. Chemical structure of a12K4 modified with different amino acids.

[0159] Figure 44. The percentage of mRNA expression in liver, spleen, and lung mediated by a12K4 modified with different amino acids.

[0160] Figure 45. The percentage of mRNA expression in liver, spleen, and lung mediated by a16K2, aat12K3, and aam12K5 modified with different amino acids.

[0161] Figure 46. Heatmap of Luc mRNA transfection efficiency of LNP formed by different PILs on MDA-MB-231 cells (normalized to ALC-0315).

[0162] Figure 47. Hit rate statistics of LNP containing different numbers of AIFA in mRNA delivery effect analysis in vitro and in vivo.

[0163] Figure 48. Three key factors determining the difference in transfection efficiency of LNP in vitro and in vivo: stability, membrane fusion ability, and surface charge.

[0164] Figure 49. mRNA release efficiency of different LNP under low concentration of Triton X-100.

[0165] Figure 50. Principle of DCVJ fluorescent probe testing LNP hydrophobic lipid moiety microviscosity and microviscosity heat map of different LNPs at 30°C.

[0166] Figure 51. Schematic diagram of LNP mouse serum stability test and mRNA release ratio heat map of different LNPs in mouse serum after 1 hour incubation.

[0167] Figure 52. Molecular dynamics simulation of hydrogen bonding of PIL / RNA complex.

[0168] Figure 53. Schematic diagram of FRET-based experiment of LNP disassembly by lysosome-like vesicle interaction and LNP disassembly percentage heat map under lysosome-like vesicle interaction.

[0169] Figure 54. Schematic diagram of LNP-mediated lysosome-like vesicle rupture experiment and LNP-mediated lysosome-like vesicle rupture percentage heat map.

[0170] Figure 55. Schematic diagram of LNP red blood cell lysis experiment and LNP-mediated red blood cell lysis percentage heat map.

[0171] Figure 56. Laser confocal microscope image of Cy5-mRNA-LNP uptake in MDA-MB-231 cells.

[0172] Figure 57. Laser confocal microscope image of Cy5-mRNA-LNP endosome escape in MDA-MB-231 cells and endosome-mRNA fluorescence co-localization analysis.

[0173] Figure 58. Schematic diagram of PILOT LNP-mediated Cre mRNA organ-targeted delivery in Ai9 mice.

[0174] Figure 59. Ex vivo imaging of mouse liver, spleen, lung, heart, kidney, etc. after receiving different Cre mRNA-PILOT LNPs.

[0175] Figure 60. Fold increase of mCherry fluorescence intensity in mouse liver, spleen, lung, heart, kidney, etc. after receiving different Cre mRNA-PILOT LNPs relative to the PBS group.

[0176] Figure 61. Analysis results of different cell positive percentage of liver-targeted PILOT LNP selectively editing liver.

[0177] Figure 62. Analysis results of different cell positive percentage of lung-targeted PILOT LNP selectively editing lung.

[0178] Figure 63. Analysis results of different cell positive percentage of spleen-targeted PILOT LNP selectively editing spleen.

[0179] Figure 64. Schematic of membrane-anchored GFP reporter cell line.

[0180] Figure 65. Lead editing efficiency at different PEmax mRNA:epegRNA mass ratios.

[0181] Figure 66. Lead editing efficiency at different total RNA doses.

[0182] Figure 67. Fluorescence imaging of tissue sections of mouse liver after PBS and a12Dab4 PILOT LNP dosing.

[0183] Figure 68. Fluorescence imaging of tissue sections of mouse lung after PBS and Am-Ka12K4 PILOT LNP dosing.

[0184] Figure 69. Next-generation sequencing shows lead editing efficiency in liver and lung.

[0185] Figure 70. PIL chemical structure modified with bone-targeting group bisphosphonate.

[0186] Figure 71. Bone-targeting in vivo data. DETAILED DESCRIPTION

[0187] Definitions

[0188] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0189] In this application, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, and / or elements, but do not preclude the presence or addition of one or more other features, steps, and / or elements. It will be further understood that when the terms “comprises” and / or “comprising” are used, that they encompass the meanings indicated by “consisting of” and “consisting essentially of.” The term “or combinations thereof’ means a combination of at least one of the listed items.

[0190] Unless otherwise indicated, all numbers used herein to describe any embodiment are to be understood, in all instances, to be modified in all instances by the term "about." The term "about" when applied to one or more values of interest is meant to encompass values that are close to or within an acceptable error range of the stated value. In one instance, the term "about" means any value that is within less than or equal to 10% of the value being modified by the term "about." Alternatively, "about" can mean within 3 or more standard deviations, within 5 times, or within 2 times, as is common practice in the art.

[0191] The term "alkyl chain" as used herein includes saturated alkyl chains and unsaturated alkyl chains, and also linear, branched, or cyclic alkyl chains. In the definition of the present application, the "alkyl chain" can contain one or more linking groups L in addition to the carbon skeleton. The term "unsaturated alkyl chain" means an alkyl chain containing one or more unsaturated bonds, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unsaturated bonds, e.g., carbon-carbon double bonds or carbon-carbon triple bonds. The term "alkyl chain" as used herein also includes optionally substituted alkyl chains, e.g., hydroxy-substituted alkyl chains.

[0192] The term "hydrocarbyl" as used herein means a group derived from a hydrocarbon molecule (a compound of carbon and hydrogen). In certain embodiments, a "hydrocarbyl" of the present application means an aliphatic hydrocarbyl group. In certain embodiments, a "hydrocarbyl" of the present application means a linear or branched alkyl, alkenyl, or alkynyl group. For example, a "saturated or unsaturated hydrocarbyl group having from 1 to 25 carbon atoms" of the present application means a linear or branched alkyl, alkenyl, or alkynyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. Representative "alkyl" groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, sec-butyl, isobutyl, t-butyl, and isopentyl. Representative "alkenyl" groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl. Representative "alkynyl" groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0193] The term "lower alkyl" as used herein means an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms, e.g., 1, 2, or 3 carbon atoms.

[0194] As used herein, the term "branched" or "branching" refers to a given structure, e.g., an alkyl chain, a hydrocarbyl group, an alkyl group, containing a tertiary carbon bonded to three other carbon atoms or a quaternary carbon atom bonded to four other carbon atoms.

[0195] As used herein, the term "optionally substituted" refers to replacement of 1 to 6 hydrogen groups in a given structure with a particular substituent group, including but not limited to hydroxyl, hydroxylalkyl, alkoxy, halogen, alkyl, nitro. Preferably, "optionally substituted" refers to "optionally hydroxyl substituted". Preferably, 0, 1, 2, or 3 hydrogen groups in an alkyl chain are replaced with a hydroxyl group.

[0196] In the present application, the symbol "-" refers to a single bond, does not indicate any preferred stereochemistry, and encompasses all stereoisomers and mixtures thereof. When drawn vertically through a bond, e.g., for The symbol represents the point of attachment of the group.

[0197] Amino acid building blocks

[0198] In the present application, the term "amino acid building block" refers to a basic monomer that is linked by amide bonds to constitute a peptide or a protein, which encompasses the amino acid building blocks of Formula I in the present application and any natural or other non-natural amino acid building blocks known in the art.

[0199] In certain embodiments, the amino acid building blocks of the present application are the amino acid building blocks of Formula I. In certain embodiments, the amino acid building blocks of the present application are the amino acid building blocks that are alkylated on the primary amine of the side chain based on Nα-fluorenylmethyloxycarbonyl (Fmoc) protection (Alkylated Ionizable Fmoc-protected Amino acid, AIFA). In certain embodiments, the amino acid building blocks of the present application are the amino acid building blocks that are alkylated on the primary amine of the side chain of lysine, ornithine, 2,4-diaminobutyric acid, or 2,3-diaminopropionic acid.

[0200] In the present application, the term "containing one or more linking groups L" refers to an alkyl chain that is interrupted or spaced apart by one or more linking groups L. For example, an alkyl chain containing one linking group L can be represented as "-hydrocarbyl or hydrogen-L-hydrocarbyl or hydrogen"; an alkyl chain containing two linking groups L can be represented as "-hydrocarbyl or hydrogen-L1-L2-hydrocarbyl or hydrogen" or "-hydrocarbyl or hydrogen-L1-hydrocarbyl-L2-hydrocarbyl or hydrogen", wherein the linking groups L or L1 and L2 are independently selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof. In certain embodiments, the alkyl chain of A1 or A2 of the present application does not contain a linking group or contains 1, 2, 3, or 4 linking groups.

[0201] In the present application, the term "saturated alkyl chain" or "a alkyl chain" refers to a linear or branched alkyl group having 4 to 25 carbon atoms and not having any unsaturated bonds. In certain embodiments, the saturated alkyl chain of the present application is an alkyl group which is not substituted by any substituent. In certain embodiments, the saturated alkyl chain of the present application is -CH2CH2(CH2) q CH2CH3, wherein q is an integer from 0 to 21.

[0202] In the present application, the term "unsaturated alkyl chain" refers to a linear or branched hydrocarbon group having 4 to 25 carbon atoms containing one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, unsaturated bonds, for example carbon-carbon double or carbon-carbon triple bonds. Unsaturated alkyl chains which can be used as A1 or A2 of the present application are known in the art, for example see R1 and R2 in WO2010054406A1.

[0203] In the present application, the term "hydroxyl-containing alkyl chain" or "e alkyl chain" refers to a linear or branched hydrocarbon group having 4 to 25 carbon atoms and being substituted by at least one hydroxyl group. In certain embodiments, the hydroxyl-containing alkyl chain of the present application is a linear or branched alkyl group having 4 to 25 carbon atoms and being substituted by at least one hydroxyl group, for example 1, 2, 3, 4, 5 or 6 hydroxyl groups. In certain embodiments, the hydroxyl-containing alkyl chain of the present application is -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 0 to 21.

[0204] The alkyl chain of A1 or A2 of the present application optionally further comprises one or more linking groups L, said linking groups L being independently selected from an amide bond, an ester bond, a disulfide bond, a kethicthiol bond, an ether bond or a combination thereof.

[0205] In certain preferred embodiments, the alkyl chain of A1 or A2 of the present application contains 0, 1, 2, 3 or 4 linking groups L. In certain preferred embodiments, said linking groups L can be biodegradable groups, for example ester bonds which are stable at physiological pH but are hydrolyzed by enzymes within tissues and cells, disulfide bonds which are sensitive to reduced intracellular environments, kethicthiol bonds which respond to ROS, etc. The effect of adding biodegradable groups to alkyl chains is known in the art, for example see WO2011153493A2, WO2013086354A1.

[0206] In the present application, the term "amide-containing alkyl chain" or "aam alkyl chain" refers to an alkyl chain containing at least one amide bond (-C(=0)-NH- or -NH-C(=0)-). In the present application, when A1or A2is an amide-containing alkyl chain, the number of carbon atoms of A1or A2refers to the number of carbon atoms of the hydrocarbon group attached after the amide bond. In certain embodiments, the amide-containing alkyl chain of the present application is -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, wherein each B1is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25, wherein L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof, and B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25. In certain embodiments, the amide-containing alkyl chain of the present application is -CH2CH2CONHCH2CH2(CH2) q CH2CH3or -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, wherein q is an integer from 0 to 21.

[0207] In the present application, the term "amide-containing alkyl chain" or "aam alkyl chain" refers to an alkyl chain containing at least one amide bond (-C(=0)-NH- or -NH-C(=0)-). In the present application, when A1or A2is an amide-containing alkyl chain, the number of carbon atoms of A1or A2refers to the number of carbon atoms of the hydrocarbon group attached after the amide bond. In certain embodiments, the amide-containing alkyl chain of the present application is -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, wherein each B1is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25, wherein L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof, and B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25. In certain embodiments, the amide-containing alkyl chain of the present application is -CH2CH2CONHCH2CH2(CH2) q CH2CH3or -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, wherein q is an integer from 0 to 21.

[0208] In the present application, the term "amide-containing alkyl chain" or "aam alkyl chain" refers to an alkyl chain containing at least one amide bond (-C(=0)-NH- or -NH-C(=0)-). In the present application, when A1or A2is an amide-containing alkyl chain, the number of carbon atoms of A1or A2refers to the number of carbon atoms of the hydrocarbon group attached after the amide bond. In certain embodiments, the amide-containing alkyl chain of the present application is -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, wherein each B1is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25, wherein L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof, and B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number of 1 to 25. In certain embodiments, the amide-containing alkyl chain of the present application is -CH2CH2CONHCH2CH2(CH2) s -B2contains at least one L that is a disulfide bond. The term "kethol thioacetal-containing alkyl chain" refers to an alkyl chain containing at least one kethol thioacetal bond (-S-C(CH3)2-S-), i.e., -(B1-L) sat least one L in B2 is a ketone thiohemiacetal bond. The term "branched alkyl chain" refers to a hydrocarbon group containing at least one branching in the alkyl chain, for example -(B1-L) s at least one B1 or B2 in B2 is a branched hydrocarbon group.

[0209] In the present application, the term "amino protecting group" refers to any chemical group that stabilizes and is readily removed from the amino group of an amino acid building block in chemical reactions such as peptide synthesis. In the present application, any amino protecting group known in the art can be used, including but not limited to alkoxycarbonyl-based amino protecting groups, acyl-based amino protecting groups, or alkyl-based amino protecting groups. Common amino protecting groups include, but are not limited to, fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), p-methoxybenzyl (PMB), benzyl (Bn), trityl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), allyloxycarbonyl (Alloc), and the like. In certain embodiments, the "amino protecting group" of the present application is Fmoc.

[0210] Peptidyl ionizable lipids

[0211] In the present application, the term "peptidyl ionizable lipid" or "PIL" refers to a peptide-based ionizable lipid, which is composed of alkylated amino acid building blocks of the present application, and optionally natural or other non-natural amino acid building blocks, linked through amide bonds.

[0212] In the present invention, the term "amino acid" includes D-type amino acids and L-type amino acids. In the present invention, the term "natural amino acid" refers to amino acids that naturally exist in organisms, including the 20 different amino acids commonly found in proteins, i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In the present invention, the term "unnatural amino acid", "non-canonical amino acid", "artificial amino acid" refers to amino acids other than the above-mentioned 20 common amino acids, which are not encoded by the genetic code in organisms, including phenylalanine derivatives, tyrosine derivatives, glutamine derivatives, alanine derivatives, cysteine derivatives, serine derivatives, and lysine derivatives, etc., such as N-ethyl beta-alanine (Neb), sarcosine (Sar), alpha, beta-diaminopropionic acid (Adp), beta-amino-N-butryic acid (aBut or Bab), beta-aminoisobutyric acid (Bai), alpha-aminoisobutyric acid (Aib), gamma-aminobutyric acid (Gab), alpha-aminobutyric acid (Anb), N-methylalanine (Nma), N-ethylglycine (Neg), allothreonine (Alt), Hse, 4-amino-3-hydroxybutyric acid (Hga), 2,4-diaminobutyric acid (Dab), hydroxyproline (Hyp), isovaline (Iva), norvaline (Nor), L-cyclopropylglycine (Cpg), N-propylglycine (Npg), homocysteine (Hcy), piperidinic acid (Pip), ornithine (Orn), tert-leucine (Tle), alloisoleucine (Ali), norleucine (Nle), 2-aminoheptanoic acid (Ahe), citrulline (Cit).

[0213] In the present invention, the term "basic amino acid" refers to amino acids having a net positive charge at neutral pH, such as lysine, arginine, histidine.

[0214] The term "acidic amino acid" refers to amino acids having a net negative charge at neutral pH, such as glutamic acid and aspartic acid.

[0215] Lipid nanoparticle

[0216] In the present invention, the term "lipid nanoparticle" refers to a particle having a nanoscale size (e.g., 1-1,000 nm) comprising one or more lipids.

[0217] In certain embodiments, the lipid nanoparticle of the present invention comprises the peptide-based ionizable lipid of the present invention, an optional helper lipid, and an optional active agent.

[0218] In certain embodiments, the lipid nanoparticles described herein can have an average particle diameter of between about 50 nm to about 200 nm, for example, about 100 nm to about 120 nm or about 150 nm to about 190 nm. In certain embodiments, the lipid nanoparticles described herein can have a surface charge of about -20 mV to about +20 mV, for example, about +3 mV to about +7 mV, about 0 mV to about 5 mV, about -3 mV to about +1 mV. In certain embodiments, the lipid nanoparticles described herein can have a polydispersity index (PDI) value of between about 0 to about 0.3, for example, about 0.1 to about 0.2. In certain embodiments, the lipid nanoparticles described herein can have an apparent acid dissociation constant of between about 5 to about 8, for example, greater than about 7.3, about 6.5 to about 7.3, about 6 to about 6.5.

[0219] In the present application, the term "ionizable lipid" or "cationic lipid" refers to a lipid that has a net positive charge at a selected pH, such as physiological pH. In certain embodiments, in addition to the peptide-based ionizable lipids of the present application, the lipid nanoparticles described herein optionally comprise additional ionizable lipids, for example, (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), (4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and the like.

[0220] The ionizable lipids or peptide-based ionizable lipids can be included in the lipid nanoparticles in different amounts. For example, the lipid nanoparticles can include about 10 mol% to about 100 mol%, for example, about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol% of the ionizable lipids or peptide-based ionizable lipids, based on total lipids.

[0221] In certain embodiments, the “active agent” described herein refers to any active substance intended to be delivered by the lipid nanoparticle, such as a therapeutic agent, an immunologically active agent, etc. In certain embodiments, the active agent described herein is a nucleic acid molecule, such as an antisense oligonucleotide (ASO), an mRNA, an siRNA, a guide RNA (gRNA), a viral vector, etc. In certain embodiments, the active agent is a gene therapy agent. In certain embodiments, the gene therapy agent is a CRISPR-based gene editing element such as a prime editing element or a chimeric antigen receptor T immunotherapy agent. In certain embodiments, the active agent is a PEmax mRNA and an engineered prime editing guide RNA (epegRNA). In certain embodiments, the mass ratio of PEmax mRNA and epegRNA in the active agent is from 10:1 to 1:10, such as from 1:2 to 2:1, or 1:1. In certain embodiments, the mass ratio of total lipids to active agent in the LNP is about 1-100:1, such as about 20-80:1, for example about 40:1. In certain embodiments, the active agent is present in the LNP at a level of about 1 ng to about 10 pg, such as about 400 ng to 1 pg, about 500 ng, or about 600 ng.

[0222] In the present application, the term “helper lipid” refers to a lipid in the lipid nanoparticle other than the cationic lipid that contributes to the stability and delivery efficiency thereof. In the present application, examples of the helper lipid include, but are not limited to, a phospholipid, a sterol, a PEG lipid, etc.

[0223] In the present application, examples of the phospholipid include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-1-carboxylate (DOPE-mal), etc. The phospholipid can be included in the lipid nanoparticle in different amounts. For example, the lipid nanoparticle can include the phospholipid at about 0 mol% to about 30 mol%, such as about 5 mol% to about 30 mol%, about 8 mol% to about 15 mol%, based on the total lipids. In certain embodiments, the lipid nanoparticle can comprise a molar ratio of the phospholipid to the peptidyl ionizable lipid of about 1:10 to about 1:20. In certain embodiments, the molar ratio is about 1:5, 2:9, 1:4, 1:2, 8:9, 1:1, 4:3, 2:1, 3:1, 4:1, 6:1, 8:1, to about 10:1.

[0224] In the present application, examples of steroids include, but are not limited to, cholesterol and its derivatives, ergosterol, lanosterol, stigmasterol, sitosterol, and the like. Among them, examples of cholesterol derivatives include, but are not limited to, 5a-cholestanol, 5 -coprostanol, cholesteryl-(2'-hydroxy)-ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5a-cholestan, cholestenone, 5a-cholestanone, 5 -cholestanone, and the like. The steroid can be included in the lipid nanoparticle in different amounts. For example, the lipid nanoparticle can include about 0 mol% to about 70 mol%, for example, about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol% of a steroid, based on total lipids. In certain embodiments, the lipid nanoparticle can comprise a molar ratio of steroid to peptidyl ionizable lipid of about 1 :4 to about 8: 1. In certain embodiments, the molar ratio is about 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, to about 8: 1, or any range derivable therein. In certain embodiments, the molar ratio is about 1 : 1 to about 6: 1, such as 2: 1 or 3: 1.

[0225] In the present application, PEG lipid refers to any complex of polyethylene glycol (PEG) and a lipid, examples of which include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and the like. In certain embodiments, the PEG lipid is PEG-modified distearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In certain embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol-methoxy(polyethylene glycol) MW 2000 (DMG-PEG2000). In certain embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In certain embodiments, the PEG modification has a molecular weight of about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. The PEG lipid can be included in the lipid nanoparticle in varying amounts. For example, the lipid nanoparticle can include about 0 mol% to about 10 mol%, e.g., about 0.01 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 1 mol% to about 2 mol% of the PEG lipid, based on total lipid. In certain embodiments, the lipid nanoparticle can comprise a molar ratio of PEG lipid to peptidyl ionizable lipid of about 1 : 1 to about 1 : 100. In certain embodiments, the molar ratio is about 1 : 1, 3:5, 1 :2, 1 :5, 1 : 10, 1 :20, 1 :30, 1 :40, 1 :50, 1 :60, 1 :70, 1 :80, 1 :90, to about 1 : 100.

[0226] In certain embodiments, the LNP of the present application is an organ or tissue-targeted LNP, e.g., a spleen-targeted LNP, a liver-targeted LNP, a lung-targeted LNP, or a bone-targeted LNP. “Targeted” or “specific” in the present application means that more than 50%, e.g., more than 70%, more preferably more than 90%, of the expression is in a particular organ or tissue when the LNP is delivered in vivo. Alternatively, “target organ targeting” in the present application means that the lipid nanoparticle is delivered to a target organ at least 25%, at least 50%, at least 75%, at least 80%, or at least 90% of the amount administered when administered in vivo. “Dual targeting” in the present application means that the lipid nanoparticle is delivered to each of two target organs at least 25%, at least 30%, at least 40%, or at least 45% of the amount administered when administered in vivo.

[0227] In certain embodiments, the LNP of the present application is a Peptide Ionizable Lipid-driven Organ Targeting lipid nanoparticle (PILOT LNP). Specifically, PILOT LNP refers to an organ-targeting, in vivo modulatable LNP mediated by PIL without additional ligand or lipid molecules.

[0228] Examples

[0229] Example 1 Construction of Fmoc group-protected alkylated ionizable amino acid building blocks (AIFA)

[0230] A series of Nα-Fmoc-protected alkylated amino acid building blocks were synthesized by reductive amination of primary amino groups with aliphatic aldehydes, ring-opening of epoxides with primary amino groups, and Michael addition of acrylamide and acrylate with primary amino groups, using Nα-Fmoc-protected amino acids with primary amino groups in side chains as substrates (Figure 2).

[0231] The specific synthetic routes are shown in Figure 3. Fmoc-L-lysine (Lys), Fmoc-L-ornithine (Orn), Fmoc-L-diaminobutyric acid (Dab), and Fmoc-L-diaminopropionic acid (Dap) were dissolved in 1,2-dichloroethane containing 1% acetic acid, and activated at room temperature for 30 minutes. Then, sodium triacetoxyborohydride was added, and the mixture was stirred at room temperature for 3 days. The reaction solution was purified by post-treatment and flash column chromatography to obtain the final product as a light yellow sticky substance. The remaining types of amino acids were synthesized using Boc-L-lysine as the starting material. Boc-L-lysine was reacted with epoxy decane, epoxy dodecane, and epoxy tetradecane in ethanol at 80°C for 3 days, with dodecyl acrylamide in dimethyl sulfoxide for 7 days, and with dodecyl acrylate in dimethyl sulfoxide for 3 days, respectively. After purification by flash column chromatography, a series of Boc-protected alkylated lysines were obtained. Then, the Boc-protected alkylated lysines were dissolved in dichloromethane, and trifluoroacetic acid was added. The mixture was stirred at room temperature for 4 hours to remove the Boc group. The alkylated lysine after removal of the Boc group was dissolved in a mixture of acetonitrile and tetrahydrofuran containing N,N-diisopropylethylamine. A solution of fluorenylmethoxycarbonyl succinimidyl in tetrahydrofuran was slowly added dropwise to the mixture, and the mixture was stirred at room temperature overnight. The reaction solution was purified by post-treatment and flash column chromatography to obtain the final product as a light yellow sticky substance. The correct synthesis of the final product was confirmed by nuclear magnetic resonance hydrogen spectrum. The chemical structure and nuclear magnetic resonance results of representative final products are shown in Figure 4.

[0232] Example 2 Construction of PIL lipid library

[0233] Based on the polypeptide solid-phase synthesis technology, the above-mentioned alkylated amino acid building blocks were used to construct a PIL lipid library containing 1-5 amino acid building blocks (Figure 5), wherein the specific chemical structure of the PIL lipids contained is shown in Figures 6-11. According to the type and number of alkylated amino acids contained in the PIL, the PIL is named as X o Y n wherein X represents the type of alkyl chain: a is a saturated alkyl chain obtained by aldehyde reduction amination, e is a hydroxyl-containing alkyl chain obtained by epoxy ring opening, aam is an amide-containing alkyl chain obtained by acrylamide Michael addition, aat is an ester-containing alkyl chain obtained by acrylate Michael addition; O represents the number of carbon atoms in the alkyl chain; Y represents the type of amino acid: Lys / K is lysine, Orn is ornithine, Dab is 2,4-diaminobutyric acid, and Dap is 2,3-diaminopropionic acid; n is the number of amino acid building blocks.

[0234] Taking a12K4 lipid as an example, which represents a 4-l lysine building block lipid containing 12 carbon atoms of saturated alkyl chain, the specific synthesis route is shown in Figure 12. Using Rink Amide resin as a solid-phase carrier, first, dichloromethane was added to the resin, and the resin was swelled at room temperature for 30 minutes; then the dichloromethane was removed, and a solution containing 20% piperidine in N,N-dimethylformamide was added to the resin to remove the Fmoc group on the surface of the resin; after removing the Fmoc, the resin was washed with N,N-dimethylformamide and dichloromethane for 3 times respectively; then a certain amount of AIFA, benzotriazol-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA) were weighed and dissolved in dichloromethane / N,N-dimethylformamide solution, and the solution was added to the resin, and the reaction was carried out at room temperature for 2 hours, and the reaction end point was detected by using ninhydrin color reaction; after the reaction was completed, the reaction solution was removed, and the resin was washed with N,N-dimethylformamide and dichloromethane for 3 times respectively; the Fmoc removal and amino acid condensation reaction were repeated until the final synthesis of a12K4 PIL. After the Fmoc removal of the last amino acid, a mixture of acetic anhydride / N,N-diisopropylethylamine / N,N-dimethylformamide (volume ratio of 1 / 1 / 1) was added to the resin, and the reaction was carried out at room temperature for 1 hour to acetylate the terminal amino group of the PIL lipid; for the PIL with free amino group at the N-terminus, the N-terminal acetylation step was omitted; the final product was obtained as a light yellow viscous liquid after being cleaved from trifluoroacetic acid and purified. The results of nuclear magnetic resonance hydrogen spectrum show that the peptide-based ionizable lipid a12K4 is correctly synthesized (Figure 13).

[0235] Example 3 Construction of a PIL-based LNP library

[0236] A lipid ethanol solution was prepared with a molar ratio of PIL:distearoylphosphatidylcholine (DSPC):cholesterol (Chol):dimyristyl polyethylene glycol (DMG-PEG) = 46.3:9.4:42.7:1.6 (FIG. 14), and a series of PIL-based Luc mRNA-LNPs (FIG. 15) were prepared by mixing the lipid ethanol solution with a sodium citrate solution (10 mM, pH 3.0) of Luc mRNA at a mass ratio of total lipid / Luc mRNA = 40:1 by microfluidization technology, and their physicochemical properties were characterized.

[0237] Physicochemical properties of PIL-based LNPs

[0238] First, the encapsulation efficiency of Luc mRNA (SEQ ID NO: 1) by LNP was calculated by determining the free mRNA concentration in the mRNA-LNP solution and the total mRNA concentration in the solution after the LNP was fully lysed with Triton X-100, respectively, using RiboGreen fluorescent dye. The results showed that the encapsulation efficiency of PIL lipids for mRNA was between 62.2% and 92.1% (FIG. 16). Among them, the encapsulation efficiencies of a6K1, a6K2, a8K1, and a12Dap5 were relatively low, between 60% and 70%; the encapsulation efficiencies of the rest of the PILs were all above 70%. This indicates that most of the PILs have a relatively high encapsulation efficiency for mRNA.

[0239] Next, the hydrated particle size, surface charge, and polydispersity coefficient of the LNP were investigated using dynamic light scattering technology (FIGS. 17-19). The inventors found that the LNP formed by the PIL containing only one AIFA had the smallest particle size, about 100 nm-120 nm; the LNP formed by the PIL containing 2-5 AIFAs had a slightly larger particle size, about 150 nm-190 nm. The particle size distribution of all the prepared LNPs was relatively uniform, and the PDI value was about 0.1-0.2. In terms of the surface charge of the LNP, the inventors found that the LNP formed by the PIL containing one AIFA had a potential of about +3 mV-+7 mV; as the number of AIFAs increased, the potential of the LNP gradually decreased, the LNP formed by the PIL containing two AIFAs had a charge of about 0 mV-+5 mV, and the LNP formed by the PIL containing 3-5 AIFAs had a charge of about -3 mV-+1 mV.

[0240] Furthermore, the apparent acid dissociation constant (pKa) of LNP was determined using the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescent probe. TNS is a negatively charged fluorescent dye molecule that hardly emits light in its free state in aqueous solution; however, LNP gradually becomes protonated and carries a positive charge as the pH of the aqueous solution decreases. TNS emitting strong fluorescence after binding to positively charged lipids, and the higher the degree of protonation of LNP, the stronger the fluorescence of TNS. Based on this, the fluorescence intensity of TNS in LNP solutions with pH values ​​of 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11 was measured, and a correlation curve was plotted between intensity and pH value (Figure 20). The pH value corresponding to when the fluorescence intensity of TNS reaches 50% of its maximum intensity is the apparent pKa value of LNP. The results (Figure 21) show that as the number of AIFAs increases, the apparent pKa value of the LNPs formed gradually decreases and then levels off. Specifically, LNPs formed from PILs containing one AIFA all have apparent pKa values ​​greater than 7.3; LNPs formed from PILs containing 2-3 AIFAs have apparent pKa values ​​around 6.5-7.3; and LNPs formed from PILs containing 4-5 AIFAs show a further decrease in apparent pKa value, which then stabilizes at around 6-6.5. The possible reason for this decrease in pKa values ​​with increasing AIFA count is that the electrostatic repulsion between positive charges within the PIL molecule leads to a decrease in the protonation ability of the tertiary amine.

[0241] To further observe the morphology and structure of LNPs, cryo-electron microscopy was used to observe a12K4 LNPs. The electron microscopy results showed (Figure 22) that a12K4 LNPs mainly contain two typical structures: 1) a regular and smooth circular structure; 2) a structure containing "cavitation".

[0242] Example 5: Efficiency of PIL LNP in delivering Luc mRNA in vivo and its structure-activity relationship

[0243] Next, the efficiency of PIL LNP in delivering Luc mRNA in vivo was examined, and its structure-activity relationship was analyzed, including the effects of AIFA quantity, side chain length, alkyl chain type and length on delivery efficiency (Figure 23).

[0244] First, in vivo testing was conducted on PIL containing 1-5 α or β alkylated lysine residues (Figure 24). In C57 mice, LNP containing 6 μg Luc mRNA was injected via the tail vein, and 6 hours later, D-fluorescein potassium salt solution was injected intraperitoneally. Bioluminescent imaging was performed on the whole body of the mice and major organs such as the liver, spleen, lungs, heart, and kidneys. The imaging results are shown in Figure 25.

[0245] The fluorescence intensity of each LNP in the liver was counted and a heatmap was made (Figure 26). Overall, the PIL with 8-14 carbon atoms alkyl chain and 3-5 AIFA showed higher luciferase expression in the liver. Further analysis found that the optimal alkyl chain length was 12 carbon atoms for both a and e type alkyl chains (Figure 27), and the optimal number of AIFA was 3 or 4 (Figure 28). Among them, a12K4 lipid showed the highest liver delivery efficiency, and the measured fluorescence intensity reached saturation at a dose of 6 pg Luc mRNA, reaching 5.38 x 10 7 (photons / s / cm 2 / sr). In addition, by comparing the PILs with the same alkyl chain length but different types, the inventors found that the delivery efficiency of a alkyl chain was significantly higher than that of e alkyl chain LNP in the PIL with 3-5 AIFA (Figure 29); while there was no significant difference in the delivery efficiency of a alkyl chain and e alkyl chain LNP in the PIL with 2 AIFA (Figure 29), and the delivery efficiency of e alkyl chain was even higher than that of a alkyl chain LNP in a12K2 and e12K2. In order to further study the effect of a and e alkyl chains on in vivo delivery efficiency, a12K4 and e12K4 were used as comparison objects, and PILs containing different numbers of a and e alkyl chains were synthesized, which were 3a1e12K4, 2a2e12K4 and 1a3e12K4 respectively. The in vivo delivery results showed (Figure 30) that there was no significant difference in liver delivery between the PILs with all a alkyl chains and the PILs with two e alkyl chains, but the delivery efficiency decreased significantly with the further increase of the number of e alkyl chains. Among them, the liver delivery efficiency of a12K4 PIL was 2.34 times that of e12K4 PIL. This result is significantly different from the conclusion reported in the existing literature (the existing literature believes that the presence of hydroxyl group in e alkyl chain can significantly improve the delivery efficiency of mRNA).

[0246] Finally, the effect of the side chain length of AIFA on in vivo delivery efficiency was investigated: the side chain lengths of lysine (K), ornithine (Orn), 2,4-diaminobutyric acid (Dab), and 2,3-diaminopropionic acid (Dap) decreased in turn, with 4, 3, 2, and 1 carbon atoms, respectively. The pKa of the primary amino group of the side chain of the four amino acids decreased with the decrease of the carbon chain length, and the pKa value of Dap was about 8.49, and the pKa values of the other three amino acids were all above 9 (Figure 31). Similarly, the in vivo delivery efficiency of PILs containing different side chain lengths was tested at a dose of 6 pg Luc mRNA (Figure 32). The results showed that Dap PIL had no obvious delivery effect in vivo, while the in vivo delivery efficiency of K, Orn, and Dab PILs was relatively close, and the in vivo delivery efficiency of a12Dab4 PIL was significantly higher than that of a12K4 PIL, with a fluorescence intensity of 7.32 x 107 (photons / s / cm 2 / sr), is 1.36 times that of a12K4.

[0247] In summary, through the in vivo delivery efficiency test of PIL-based LNP, the inventors found that the number of AIFA contained in PIL, the type and length of alkyl chain, and the length of side chain all have important influence on the efficiency of mRNA delivery. Among them, the PIL with optimal delivery efficiency is characterized by 3-5 AIFA, a side chain of 2-4 carbon atoms, a saturated alkyl chain, and an alkyl chain of 10-14 carbon atoms, and the optimal lipid is a12Dab4.

[0248] Example 6 Relationship between PIL structure and organ-targeted mRNA delivery

[0249] Further, the relationship between the PIL structure and organ-targeted mRNA delivery was explored (Figure 33). First, the proportion of fluorescence intensity of different PILs in the liver and spleen was compared (Figure 34), in which the PILs with 4-5 AIFA, and 3 AIFA and an alkyl chain of 10-14 carbon atoms had a liver fluorescence intensity proportion of more than 80%, showing good liver-targeted delivery efficiency; while the PILs with 2 AIFA and an alkyl chain of 16 and 18 carbon atoms (a16K2 and a18K2) had a spleen fluorescence intensity proportion of more than 90%, showing excellent spleen-specific delivery efficiency; the liver / spleen ratio of the remaining types of PILs was between 25-75%, indicating that they had close mRNA delivery efficiency to the two main organs of liver and spleen.

[0250] Next, the relationship between alkyl chain type and targeting was further analyzed. In the 12-carbon alkyl chain PILs, the expression percentages of different alkyl chain types containing 1-5 AIFAs in the liver, spleen, and lung were compared (Figure 35). The inventors found that as the number of AIFAs increased from 1 to 5, the percentages of delivered mRNA in each organ and their trends varied (Figure 36). Among them, the percentage of a alkyl chain gradually decreased in the spleen and lung, but gradually increased in the liver, reaching 95% when the number of AIFAs increased to 5; the percentage of e alkyl chain in the liver showed a trend of first increasing and then decreasing, reaching a maximum (more than 90%) when the number of AIFAs was 3, while the trend in the spleen was opposite, first decreasing and then gradually increasing, also reaching a minimum when the number of AIFAs was 3, and the percentage in the lung did not change significantly, all being relatively low (<5%); the percentage of aam alkyl chain in the spleen gradually increased to more than 80%, and the percentage in the lung also increased slightly, while the percentage in the liver gradually decreased to less than 10%; the percentage of aat alkyl chain in the liver and spleen did not change significantly, and the liver / spleen ratio remained around 1 / 1. These results indicate that the choice of alkyl type is crucial for the development of PILs targeting different organs. For liver-targeting LNP, PILs containing a and e alkyl chains with 3-5 AIFAs should be selected; for spleen-targeting LNP, PILs containing aam alkyl chains with 3-5 AIFAs should be selected; and for liver and spleen dual-targeting LNP, PILs containing aat alkyl chains can be selected.

[0251] Next, the effects of C-terminal and N-terminal group types on the targeting and expression efficiency of delivered mRNA were investigated. As described previously, the use of the solid-phase synthesis support Rink Amide resin can produce PILs with C-terminal amide. Here, another solid-phase synthesis support, 2-chlorotrityl chloride (2-CTC) resin, was introduced to produce PILs with a free carboxyl group at the C-terminus (Figure 37). As described previously, PILs with a free amino group at the N-terminus were also prepared by omitting the step of N-terminal amino acetylation during synthesis. Taking the original N-terminal acetylated and C-terminal amide a12K2 and a12K4 as examples, Am-a12K2 and Am-a12K4 represent lipids with a free amino group at the N-terminus and a C-terminal amide; a12K2-Ca and a12K4-Ca represent lipids with N-terminal acetylation and a free carboxyl group at the C-terminus; and Am-a12K2-Ca and Am-a12K4-Ca represent lipids with a free amino group at the N-terminus and a free carboxyl group at the C-terminus (Figure 38). By comparing the above PILs with different terminal groups but the same peptide backbone, it was found that the introduction of a single free amino or carboxyl group increased the expression efficiency of mRNA delivered by PILs in the spleen; when both amino and carboxyl groups were introduced, the phenomenon of spleen specificity enhancement disappeared, and the liver and spleen percentages were close to those of the original PIL (Figure 39).

[0252] The above results show that different end groups are closely related to mRNA organ-targeted delivery, so further study was conducted on the effect of adding different natural amino acids to the end of PIL on mRNA in vivo delivery. First, 20 natural amino acids were used to modify the N-terminus of a12K2, and the specific PIL structure is shown in FIG. 40.

[0253] The in vivo delivery results show (FIG. 41) that in the N-acetylated a12K2, the N-histidine-modified Ha12K2 PIL significantly enhances the specificity (>90%) and intensity of liver mRNA expression; and the N-lysine- and arginine-modified Ka12K2 and Ra12K2 PILs can specifically deliver mRNA to the spleen (accounting for >90%). Further exploration of PILs with free amino groups at the N-terminus and different numbers of basic amino acids at the N-terminus, including Am-Ka12K2, Am-Ra12K2, Am-K2a12K2, Am-R2a12K2, Am-K3a12K2, Am-R3a12K2, found that the N-terminal free amino group can significantly increase the mRNA delivery of the original spleen-targeted Ka12K2 and Ra12K2 in the lungs, and the lung specificity and intensity gradually increase with the increase in the number of basic amino acids. Similarly, PILs with free carboxyl groups at the C-terminus and different numbers of acidic amino acids at the C-terminus (including a12K2E-Ca, a12K2E2-Ca, a12K2E3-Ca, a12K2D-Ca, a12K2D2-Ca, a12K2D3-Ca) can selectively deliver mRNA to the spleen (accounting for >90%), but the intensity decreases with the increase in the number of acidic amino acids.

[0254] In addition, it was found that the cysteine-modified Ca12K2 PIL greatly improved the efficiency of liver-targeted mRNA delivery, with a liver fluorescence intensity of about 35 times that of a12K2; the proline- and tryptophan-modified Pa12K2 and Wa12K2 PILs significantly enhanced the specificity and intensity of spleen mRNA expression; the tyrosine- and phenylalanine-modified Ya12K2 and Fa12K2 PILs also significantly improved the specificity and intensity of liver mRNA expression; the delivery efficiency of other amino acid-modified PILs varied, but there was no significant difference in the overall mRNA expression ratio of each organ (FIG. 42).

[0255] To further verify the universality of the above amino acid modification in changing the organ-specific expression of mRNA, a12K4, which has stronger liver targeting and higher efficiency, was selected as the object and similar amino acid modification was performed. The specific structure is shown in Figure 43. Similarly, it was observed that after 1 basic amino acid was modified at the N-terminus of acetylated a12K4 (Ka12K4 and Ra12K4), the specificity of its mRNA expression in the spleen was significantly enhanced (>90%). PILs with free amino groups at the N-terminus and different numbers of basic amino acids, including Am-Ka12K4, Am-Ra12K4, Am-K2a12K4, Am-R2a12K4, Am-K3a12K4, and Am-R3a12K4, could increase the specific expression of mRNA in the lungs. The specificity of PILs with different numbers of lysine modified at the N-terminus in the lungs was all >90%. The specificity and intensity of PILs with different numbers of arginine modified at the N-terminus in the lungs were both enhanced with the increase in the number of arginine. In the modification of acidic amino acids, it was also observed that PILs with free carboxyl groups at the C-terminus and different numbers of acidic amino acids modified at the C-terminus, including a12K4E-Ca, a12K4E2-Ca, a12K4E3-Ca, a12K4D-Ca, a12K4D2-Ca, and a12K4D3-Ca, could selectively deliver mRNA to the spleen (more than 90%). The intensity gradually decreased with the increase in the number of acidic amino acids (Figure 44).

[0256] To further verify the universality of the amino acid modification strategy, PILs with different initial mRNA expression levels in various organs, different numbers of AIFAs, and different types of AIFAs were selected, and the mRNA delivery efficiency and the proportion of each organ after modification were analyzed. As shown in Figure 45, the liver target proportion of a16K2, which had an initial spleen target proportion of >90%, reached >90% after modification with histidine (Ha16K2) and cysteine (Ca16K2), and the lung target proportion was >90% after modification with two lysines; the liver target, lung target, and spleen target proportions of aat12K3, which had an initial liver and spleen mRNA expression proportion of about 1:1, reached >90% after modification with histidine, lysine, and glutamic acid; the liver target and lung target proportions of aam12K5, which had an initial spleen target proportion of >80%, were both >85% after modification with histidine, lysine, and arginine. At the same time, it was also found that the initial expression intensity of PIL in the liver directly determined the delivery efficiency of the amino acid modified PIL in the spleen and lungs, such as the fluorescence intensity values of a12K4 PIL in the spleen and lungs were significantly higher than those of a12K2 PIL lipid. This PIL-mediated, organ-selective and controllable LNP without the need for any additional ligands and lipid molecules added to the LNP was called PILOT LNP.

[0257] According to the experimental results, the inventors summarized a general amino acid modification method for regulating the organ-specific delivery of PIL by PILOT LNP, including: 1) histidine, cysteine, tyrosine and phenylalanine modified PIL enhances mRNA liver-specific expression and intensity; 2) proline and tryptophan modified PIL enhances mRNA spleen-specific expression; 3) single basic amino acid modification and N-terminal acetylation of PIL enhances mRNA spleen-specific expression; 4) 1 to multiple basic amino acid modification and N-terminal free amino group of PIL enhances mRNA lung-specific expression; 5) 1 to multiple acid amino acid modification of PIL enhances mRNA spleen-specific expression. This general amino acid modification strategy will provide certain guidance for the design of a new generation of ionizable lipids with mRNA organ-selective delivery capability, and the constructed PIL lipid solid-phase synthesis platform is expected to further improve the synthesis and screening efficiency of organ-selective ionizable lipids under the guidance of this strategy.

[0258] Example 7 Mechanism study of the relationship between PIL structure and mRNA delivery efficiency and specificity

[0259] To further understand the relationship between PIL structure and mRNA delivery efficiency and specificity, a series of mechanism-based studies were carried out. First, the in vitro delivery efficiency of LNP was tested on human breast cancer cells MDA-MB-231, and ALC-0315 was used as a standard to normalize the delivery efficiency of each LNP. The processing results show (Figure 46) that PIL containing 1-2 AIFA performs much better than PIL containing 4-5 AIFA which performs better in vivo in vitro transfection, while PIL containing 3 AIFA exhibits good delivery efficiency in vivo and in vitro. To better quantify this in vitro-in vivo difference, the cell level transfection efficiency of ALC-0315, and the liver intensity of mice reaching 5x10 6 (photons / s / cm 2 / sr) as the respective baseline, the hit rate was calculated. The statistical results show (Figure 47) that in cell transfection, the hit rates of PIL containing 1 and 2 AIFA are 60% and 86.7%, respectively, which are much higher than those of PIL containing 4 (20%) and 5 (6.7%) AIFA; while in mice, the hit rates of PIL containing 1 and 2 AIFA are 0% and 6.7%, respectively, which are much lower than those of PIL containing 4 (60%) and 5 (33.3%) AIFA; PIL containing 3 AIFA exhibits high hit rates in vitro and in vivo, which are 73.3% and 53.3%, respectively.

[0260] To better understand the reason for the difference in LNP delivery efficiency in vitro and in vivo, the fate of LNP in vitro and in vivo was briefly analyzed. Generally, the transfection ability of LNP is closely related to its stability, serum protein crown, cell uptake ability, endosome escape, etc. Based on this, it is speculated that the surface charge and membrane fusion ability of LNP are the key factors that determine the difference in delivery efficiency in vitro and in vivo (Figure 48).

[0261] To this end, they were investigated one by one. First, the stability of LNP was evaluated. LNP was incubated with 1% and 2% (which can completely destroy the LNP structure and fully release mRNA) Triton X-100 solution, and the stability of LNP was evaluated by comparing the release amount of mRNA in different concentrations of Triton X-100 solution (Figure 49). The test results show that the LNP formed by the PIL containing 4 and 5 AIFA and the alkyl chain length of 10-18 carbons does not completely release the mRNA it carries in 1% concentration of Triton X-100 solution. This indicates that these lipids have not been completely destroyed by low-concentration Triton X-100, and the LNP formed by them has stronger stability.

[0262] Further, the reason for this difference in stability was explored using the 9-(2,2-dicyanovinyl) guanidinium (DCVJ) fluorescent probe. The DCVJ molecule is a fluorescent molecular rotor, and its fluorescence characteristics are greatly affected by the angle of rotation of the single bond between its aromatic ring and olefin group. In a low-viscosity local microenvironment, its single bond rotation is easy to occur, which in turn promotes the opening of its excited state non-radiative transition channel, and the fluorescence is quenched; while in a high-viscosity microenvironment, its molecular rotation is limited, and the non-radiative transition is blocked, resulting in an increase in fluorescence (Figure 50). Using this characteristic of the DCVJ probe, the local microviscosity of the hydrophobic structure of LNP was tested. The results show that with the increase of the number of AIFA and the length of the alkyl chain, the microviscosity of the hydrophobic structure of LNP gradually increases. The reason for this phenomenon is that the lipids containing more AIFA, the local arrangement of AIFA monomers is more compact, and the hydrophobic tail is more close in space, which makes the local activity of hydrophobic alkyl chain limited, and the viscosity increases; for the same reason, the lipids containing long alkyl chains have lower activity than the lipids containing short alkyl chains. The synergistic effect of the above two points eventually makes the LNP containing more AIFA (such as 4-5) and longer alkyl chain (10-18) exhibit stronger stability.

[0263] The stage from the intravenous injection of LNP into the blood circulation to the endocytosis by specific cells requires the maximum protection of mRNA from degradation and premature release. To this end, the stability of LNP in mouse serum and the release of mRNA were further evaluated (Figure 51). The results showed that after incubation with mouse serum at 37°C for 1 hour, no premature release of mRNA (<1%) occurred for LNP formed by PIL containing 3-5 AIFA; while different degrees of premature release of mRNA occurred for LNP formed by PIL containing 1-2 AIFA, especially for LNP containing 1 AIFA, the content of prematurely released mRNA was between 4.46%-19.81%. From the above results, the requirement for LNP stability in vivo is higher than that in vitro, which explains why the in vivo delivery effect of LNP containing 3-5 AIFA is much better than that of LNP containing 1-2 AIFA. However, if LNP is too stable, it may not be able to completely release mRNA in the cell, which will also hinder the delivery efficiency of mRNA.

[0264] The inventors speculate that the reason for the observed decrease in the in vivo delivery efficiency of LNP with increasing number of e-alkyl chains may be that the additional hydroxyl groups provided by the e-alkyl chains form more hydrogen bonds with mRNA, which in turn makes the mRNA binding more firm and not easy to release. To verify this guess, molecular dynamics simulation was performed on the process of hydrogen bonding between a12K2, e12K2, a12K4, 2a2e12K4, e12K4 and 20nt RNA. The simulation results show (Figure 52) that after the a12K2 / RNA complex reaches stability, the number of hydrogen bonds formed is 3, while the hydroxyl group of e12K2 provides additional hydrogen bonds for the e12K2 / RNA complex, and the number of hydrogen bonds reaches 7 after the complex stabilizes; for a12K2 LNP which is not stable enough in vivo, the introduction of additional hydroxyl groups improves the stability of LNP and enhances the in vivo delivery efficiency of LNP. Similarly, it is observed that 2a2e12K4 and e12K4 can form up to 13 hydrogen bonds throughout the simulation process, which is higher than the maximum of 8 hydrogen bonds of a12K4; for a12K4 LNP which is already stable enough in vivo, further stabilizing LNP through more hydrogen bonds actually reduces its in vivo delivery efficiency. In summary, the inventors found that the stability of LNP is an important factor affecting the difference in mRNA delivery efficiency of LNP in vitro and in vivo. The in vitro cell culture environment is relatively mild compared to in vivo, and the requirement for LNP stability is not high, those LNP which are more likely to release mRNA will have better transfection effect in vitro; while LNP in vivo faces a more complex environment, it needs to resist the degradation of various enzymes in blood and adsorb plasma proteins, so it requires LNP to have stronger stability, however, LNP that is too stable may not be conducive to the effective release of mRNA, therefore, it may be necessary to fine-tune the structure of the lipid to maximize the delivery efficiency.

[0265] Next, the membrane fusion ability of LNP was investigated to simulate the endosome escape efficiency. The membrane fusion ability of LNP at endosome pH (e.g. pH 5.5) can reflect its ability to escape from endosome after endocytosis to some extent. The fluorescence resonance energy transfer (FRET) assay based on 7-nitro-2, 1, 3-benzoxadiazole-labeled lipid (NBD-PE) and lissamine rhodamine B-labeled lipid (Rho-PE) was used to test the membrane fusion ability of LNP. NBD-PE and Rho-PE were incorporated into the total lipid solution to prepare NBD-PE and Rho-PE containing LNP, in which the fluorescence emitted by NBD was attenuated due to FRET to Rho molecules. In the solution at pH 5.5, LNP interacted with the artificial lysosome vesicle to disintegrate, and the FRET phenomenon disappeared, and the NBD fluorescence increased (Figure 53). The test results showed that the a and e alkyl chain LNP containing 8-14 carbon atoms had better membrane fusion ability, and the membrane fusion ability was enhanced with the increase of the number of AIFA.

[0266] Similarly, NBD-PE and Rho-PE were incorporated into the artificial lysosome vesicle, and it was also found that the a and e alkyl chain LNP containing 8-14 carbon atoms had better membrane fusion ability (Figure 54). To further verify this finding, the erythrocyte lysis ability of different LNP under pH 5.5 was evaluated (Figure 55). The erythrocyte lysis results were similar to the membrane fusion experiment results, and the a and e alkyl chain LNP containing 8-14 carbon atoms had the strongest erythrocyte lysis ability, and the LNP containing too short (6 carbon atoms) or too long alkyl chain (16 and 18 carbon atoms), aam and aat alkyl chain, and 2, 3-diaminopropionic acid had poor erythrocyte lysis ability. The results of this series of experiments were consistent with the mRNA delivery efficiency in vitro and in vivo, that is, whether for in vitro transfection or in vivo delivery, the lipid with stronger membrane fusion ability and erythrocyte lysis ability under endosome pH conditions had stronger mRNA delivery efficiency.

[0267] Finally, the effect of LNP surface charge was evaluated. A series of lipids containing a12 alkyl chain were selected for the study, which can form a series of LNP with surface charge from high to low, including: Am-K3a12K4 LNP, Am-Ka12K4 LNP, a12K1 LNP, a12K2 LNP, a12K3 LNP, a12K4 LNP and a12K5 LNP. Cy5 dye was used to label and trace mRNA, and CLSM was used to observe mRNA-LNP transfected MDA-MB-231 cells after 4 hours. The results showed (Figure 56) that the intracellular Cy5 intensity of three charge-neutral LNP (a12K3 LNP, a12K4 LNP and a12K5 LNP) was low, and only weak red fluorescence was observed, which indicated that charge-neutral LNP was less taken up by cells. In contrast, the intracellular Cy5 fluorescence observed for LNP with weak positive charge (a12K1 LNP and a12K2) was stronger than the aforementioned three LNP. Am-Ka12K4 and Am-K3a12K4 LNP with additional lysine benefited from their strong positive surface charge properties, mediating the most LNP endocytosis. In particular, Am-K3a12K4 LNP, the red fluorescence of which was observed to fill almost the entire cytoplasm under CLSM. This stronger endocytosis ability explained the stronger mRNA delivery efficiency exhibited by a12K1, a12K2, Am-Ka12K4 and Am-K3a12K4 LNP in in vitro transfection experiments. In addition, the inventors found that although Am-K3a12K4 LNP had the highest endocytosis efficiency, its in vitro transfection efficiency was not the strongest.

[0268] To this end, the endosomal escape ability of a12K4, Am-Ka12K4 and Am-K3a12K4 LNP was further evaluated. The intracellular endosome was labeled by Lysotracker Green, and the mRNA was also located using Cy5 dye (Figure 57). After 4 hours of mRNA-LNP transfection, it was observed that although a12K4 LNP was taken up by cells the least, most of the endocytosed LNP successfully escaped from the endosome, with a Pearson correlation coefficient of 0.137 between endosome and mRNA. While the escape ability of Am-Ka12K4 LNP was slightly worse than that of a12K4, it could also mediate sufficient endosomal escape (Pearson correlation coefficient of 0.340), and because of the high endocytosis efficiency, it achieved a stronger in vitro transfection effect than a12K4. Although Am-K3a12K4 LNP had the highest endocytosis efficiency, most of the endocytosed LNP could not escape from the endosome (Pearson correlation coefficient of 0.762), and thus it did not significantly improve the transfection effect compared to Am-Ka12K4 LNP. The above experimental results showed that for in vitro transfection, the cell uptake and endosomal escape efficiency of LNP were two key factors determining its transfection effect, and the surface charge of LNP had a decisive influence on these two factors. The near-neutral charge LNP had a very low content of being taken up by cells in vitro, so even if it had a strong endosomal escape ability, its in vitro transfection effect was poor. In in vivo delivery, the ApoE protein adsorbed on its surface would guide it to the liver and mediate its efficient endocytosis by hepatocytes, solving the bottleneck of low endocytosis efficiency in the first step of in vitro transfection. The positively charged LNP (such as PIL containing 1 or 2 AIFA and PIL containing lysine) could more easily adsorb on the surface of the negatively charged cell membrane in in vitro transfection, mediate more LNP endocytosis by cells, and ultimately achieve a better mRNA transfection effect. However, too much positive charge carried by lipids would reduce its endosomal escape ability, so it is necessary to balance endocytosis and endosomal escape by adjusting the charge to maximize the in vitro transfection effect. In in vivo delivery, strong positive charge often leads to the adsorption of a protein crown different from that of neutral charge LNP, resulting in its targeting of extrahepatic tissues such as spleen and lung. In summary, the three key indicators of LNP stability, endosomal escape ability and surface charge have different degrees of influence on the various stages of in vivo and in vitro delivery experienced by LNP, to some extent, explaining the structure-activity relationship of PIL and the mechanism of organ-specific delivery of PILOT LNP, and answering the question of why the in vitro and in vivo results are difficult to correspond when screening liver-targeting ionizable lipids based on in vitro transfection effect.

[0269] Example 8 PILOT LNP for functional mRNA delivery

[0270] Subsequently, the application potential of PILOT LNP in functional mRNA delivery, especially in the in vivo targeted delivery of gene editing elements, was further verified. For this purpose, one PILOT LNP each for liver (a12Dab4), spleen (a12K4E-Ca), and lung (Am-Ka12K4) targeting was selected, and a Cre-LoxP mouse (Ai9 mouse) model was used to evaluate the gene editing efficiency in the target organs by organ-targeted delivery of Cre recombinase mRNA (Cre mRNA, SEQ ID NO: 2) (FIG. 58). The Ai9 mice were given PILOT LNP containing 6 pg of Cre mRNA via tail vein injection, and 48 hours later, the liver, spleen, lung, heart, kidney, and other major organs were imaged ex vivo. The imaging results showed (FIG. 59) that compared with the PBS group, the liver-, spleen-, and lung-targeted PILOT LNP could all mediate specific Cre editing of Cre in the respective target organs. Among them, the liver mCherry intensity of the mouse receiving liver-targeted PILOT LNP was about 257 times that of the PBS group; the lung mCherry intensity of the mouse receiving lung-targeted PILOT LNP was about 28 times that of the PBS group; and the spleen mCherry intensity of the mouse receiving spleen-targeted PILOT LNP was about 4 times that of the PBS group (FIG. 60). Further, the cell types transfected by liver-, lung-, and spleen-targeted PILOT LNP in the liver, lung, and spleen were sorted. The results showed (FIGS. 61-63) that in the liver of the mouse receiving liver-targeted PILOT LNP, 74.5% of the liver parenchymal cells, 42.4% of the liver macrophages, and 22.0% of the liver sinus endothelial cells were successfully edited by Cre; in the lung of the mouse receiving lung-targeted PILOT LNP, 39.5% of the epithelial cells, 66.9% of the endothelial cells, and 10.1% of the immune cells were successfully edited by Cre; and in the spleen of the mouse receiving spleen-targeted PILOT LNP, 5.7% of the T cells, 1.6% of the B cells, 26.2% of the macrophages, and 7.3% of the NK cells were successfully edited by Cre. The above results showed that the PILOT LNP developed by the present application could specifically deliver mRNA encoding the gene editing protein Cre to different target organs such as the liver, spleen, and lung, and successfully edit specific cells in the target organs.

[0271] After verifying that PILOT LNP can successfully deliver gene editing protein Cre mRNA to specific target organs in vivo, further evaluation was made on the in vitro and in vivo delivery of prime editor (PE) based on CRISPR gene editing system. The a14K2 PIL with the best mRNA transfection effect in vitro was selected to encapsulate mRNA encoding PEmax (SEQ ID NO: 3) and engineered prime editing guide RNA (epegRNA, SEQ ID NO: 4), and its editing effect was tested in a membrane-anchored green fluorescent protein reporter cell line. In this reporter cell line, the gene encoding green fluorescent protein contains a stop codon TAG upstream, and the green fluorescent protein cannot be normally expressed; after successful correction by prime editor, the stop codon TAG is changed to CAG, and the green fluorescent protein recovers expression and is anchored to the cell membrane (as shown in FIG. 64).

[0272] First, the ratio of PEmax mRNA and epegRNA was preliminarily screened (the total input amount of RNA was fixed at 400 ng). The results showed (FIG. 65) that at the PEmax mRNA:epegRNA mass ratio of 1:2, 1:1 and 2:1, the PE editing efficiency did not have significant difference, all around 14%-16%; in addition, a14K2 LNP showed comparable transfection efficiency with commercial transfection reagent Lipofectamine TM MessengerMAX TM Further, fixing the PEmax mRNA:epegRNA mass ratio at 1:2 and increasing the amount of RNA, it was found that the PE editing efficiency increased with the increase of RNA dose, showing a certain dose dependence, and the PE editing efficiency in the highest dose group (600 ng RNA) could reach 19% (FIG. 66).

[0273] Finally, the a12Dab4 with the highest liver targeting efficiency and the Am-Ka12K4 with the lung targeting efficiency were selected to verify the in vivo delivery of PEmax mRNA and epegRNA. On the membrane-anchored green fluorescent protein transgenic mice, a12Dab4 and Am-Ka12K4 PILOT LNP containing 26.7 μg PEmax mRNA and 53.3 μg epegRNA were injected into the tail vein every 48 h for a total of three times. One week after the last injection, the liver and lung of the mice were taken and subjected to histological sectioning and fluorescence imaging. As shown in Figures 67-68, compared with the PBS control group, the liver of the mice injected with a12Dab4 PILOT LNP and the lung of the mice injected with Am-Ka12K4 PILOT LNP showed extensive and obvious fluorescence signals. The results of second-generation sequencing (Figure 69) showed that the liver and lung had a guide editing efficiency of 13.34% and 7.37%, respectively. The above results showed that the PILOT LNP developed by the present application can effectively deliver the mRNA encoding the guide editor and the epegRNA to the specific organs and successfully edit the target gene in vivo, further demonstrating its transformation potential in gene editing delivery vectors.

[0274] Example 9 Construction and in vivo verification of bone-targeting PILOT LNP

[0275] Bone-targeting small molecule alendronic acid was modified at the N-terminus of PIL lipids to achieve in vivo bone-targeting delivery of PILOT LNP. First, alendronic acid was subjected to anhydride ring-opening reaction with succinic anhydride to prepare succinylated alendronic acid; then the succinylated alendronic acid was used as a solid-phase synthesis building block to introduce at the N-terminus of a12K2 and a12K4 lipids, to synthesize alendronic acid-modified lipids Biphos-a12K2 and Biphos-a12K4 (Figure 70). Lipid ethanol solutions were prepared with a molar ratio of PIL:distearoylphosphatidylcholine (DSPC):cholesterol (Chol):dimyristyl polyethylene glycol (DMG-PEG) = 46.3:9.4:42.7:1.6 (Figure 14), and Luc mRNA-LNP based on alendronic acid-modified PIL was prepared by mixing the lipid ethanol solution with a sodium citrate solution (10 mM, pH 3.0) of Luc mRNA at a mass ratio of total lipid / Luc mRNA = 40:1 through microfluidic technology, and mice were injected with mRNA at a dose of 0.3 mpk via tail vein. After 6 h, the bilateral femur and tibia of the mice were isolated and subjected to ex vivo imaging. The imaging results showed (Figure 71) that alendronic acid-modified PIL significantly increased the expression of mRNA in the femur and tibia of mice compared with unmodified PIL. Among them, the luciferase expression in the Biphos-a12K2 group was 4.08 times that in the a12K2 group, and the luciferase expression in the Biphos-a12K4 group was 3.75 times that in the a12K4 group. The above results showed that modification of bone-targeting small molecule alendronic acid at the N-terminus of PIL could significantly enhance the expression of mRNA-LNP in the bone tissue of mice, further verifying that modification of other non-amino acid functional small molecules could enable mRNA-LNP to target other organs in addition to liver, spleen and lung.

Claims

1. An amino acid molecular building block having the structure of Formula I, ###0001### Formula I wherein m is an integer from 0 to 10, preferably from 1 to 4, A1and A2are hydrophobic tails and are independently from each other an optionally substituted saturated or unsaturated, linear or branched alkyl chain having a carbon number from 4 to 25, wherein the alkyl chain optionally contains one or more linking groups L selected from an amide bond, an ester bond, a disulfide bond, a ketholethiol bond, an ether bond, or a combination thereof, A3is an amino protecting group, and A4is a hydrogen atom or an alkyl group.

2. The amino acid molecular building block of claim 1, wherein Ai and A2 are independently of each other -(Bi-L) s B2, wherein s is an integer from 0 to 6, wherein Bi is independently in each (Bi-L) unit either absent or an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25, L is independently in each (Bi-L) unit either absent or a linking group selected from an amide bond, an ester bond, a disulfide bond, a kethol thioacetal bond, an ether bond, or a combination thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25.

3. The amino acid molecular building block according to claim 1 or 2, wherein A1and A2are independently from each other -B1-L-B2, -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, -B1-COO-B1-L-B2, or -B1-OOC-B1-L-B2, wherein each B1is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25, wherein L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a ketholethiol bond, an ether bond, or a combination thereof, and B2is an optionally substituted saturated or unsaturated hydrocarbon group having a carbon number from 1 to 25.

4. The amino acid molecular building block according to any one of claims 1 to 3, wherein A1and A2are independently from each other selected from the group consisting of a saturated alkyl chain, an unsaturated alkyl chain, a hydroxyl group containing alkyl chain, an amide bond containing alkyl chain, an ester bond containing alkyl chain, a disulfide bond containing alkyl chain, a ketholethiol containing alkyl chain, a branched alkyl chain.

5. The amino acid molecular building block according to any one of claims 1 to 4, wherein A1 and A2 are independently from each other selected from the group consisting of: -CH2CH2(CH2) q CH2CH3, -CH2CHOH(CH2) q CH2CH3, -CH2CH2CONHCH2CH2(CH2) q CH2CH3, -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, -CH2CH2COOCH2CH2(CH2) q CH2CH3, -CH2CH2OOCCH2CH2(CH2) q CH2CH3, -CH2CH2OOCCH2CH2(CH2) wherein q is an integer from 0 to 21.

6. A peptide-based ionizable lipid (PIL) having the structure of Formula II, wherein A4is as defined in any one of claims 1 to 5, n is an integer from 1 to 30, X is O or S, R1represents the N-terminus of the PIL and R1is a hydrogen atom or has an acetyl, amino acid and / or other functional group modification, R3represents the C-terminus of the PIL and R3is a hydroxyl group or has an amino, amino acid and / or other functional group modification. R2represents a side group of the PIL, R2independently at each occurrence is a side group of the amino acid building block according to any one of claims 1 to 5 a natural amino acid side group or an unnatural amino acid side group, and at least one R2in the PIL is 7. The PIL according to claim 6, wherein A1and A2are alkyl chains having a carbon number from 8 to 14 and n is from 3 to 5, preferably A1and A2are alkyl chains having a carbon number of 12 and n is 3 or 4, further preferably n is from 3 to 5, m is from 2 to 4 and A1and A2are saturated alkyl chains having a carbon number from 10 to 14, more preferably n is 4, m is 2 and A1and A2are saturated alkyl chains having a carbon number of 12.

8. The PIL according to claim 6 or 7, wherein: (i) n is from 4 to 5, (ii) n is 3 and A1and A2are alkyl chains having a carbon number from 10 to 14, (iii) n is 2 and A1and A2are alkyl chains having a carbon number from 16 to 18, (iv) A1and A2are saturated alkyl chains or hydroxyl group containing alkyl chains having a carbon number of 12 and n is from 3 to 5, (v) wherein A1and A2are amide bond containing alkyl chains having a carbon number of 12 and n is from 3 to 5, or (vi) A1and A2are ester bond containing alkyl chains having a carbon number of 12.

9. The PIL according to any one of claims 6 to 8, wherein: (a) the PIL has a basic amino acid modification, preferably a basic amino acid modification at the N-terminus and an acetylation modification. ​ (b) the PIL has a basic amino acid modification and a free amino group at the N-terminus; (c) the PIL has a proline modification, preferably a proline modification and a free amino group at the N-terminus; (d) the PIL has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof, preferably an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof and an acetylation modification at the N-terminus; (e) the PIL has a tryptophan modification, preferably a tryptophan modification and an acetylation modification at the N-terminus; (f) the PIL has an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine, or a combination thereof, preferably an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine, or a combination thereof and an acetylation modification at the N-terminus; (g) the PIL has an acidic amino acid modification, preferably an acidic amino acid modification and a free carboxyl group at the C-terminus; or (h) the PIL has a phosphorylation modification, for example a bisphosphonate modification, preferably an alendronic acid modification, at the N-terminus or the C-terminus.

10. The PIL according to any one of claims 6-9, wherein the PIL is selected from the group consisting of:

11. A method of synthesizing a PIL, the method comprising: (A) coupling a solid-phase synthesis support to an amino acid building block and removing an amino protecting group, and (B) adding an amino acid building block to a free amino group and removing an amino protecting group, repeating this step to elongate the PIL chain, wherein one or more of the amino acid building blocks in step (A) or (B) is an amino acid building block according to any one of claims 1 to 5, (C) optionally performing acetylation, and (D) cleaving the synthesized PIL chain from the solid-phase synthesis support.

12. A library of PILs comprising a plurality of PILs according to any one of claims 6-10.

13. A lipid nanoparticle (LNP) comprising a peptide-based ionizable lipid according to any one of claims 6-10, optionally a helper lipid, and optionally an active agent.

14. The LNP of claim 13, wherein the helper lipid comprises a phospholipid, a sterol, and / or a PEG lipid.

15. The LNP of claim 13 or 14, wherein the lipid nanoparticle comprises about 10 mol% to about 100 mol% of the peptide-based ionizable lipid, about 0 mol% to about 30 mol% of the phospholipid, about 0 mol% to about 70 mol% of the sterol, and / or about 0 mol% to about 10 mol% of the PEG lipid, based on total lipids.

16. The LNP of any one of claims 13-15, wherein the LNP is an organ-targeting LNP, wherein the target organ is selected from the group consisting of: lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin, preferably the LNP is a spleen-targeting LNP, a liver-targeting LNP, a lung-targeting LNP, or a bone-targeting LNP.

17. The LNP of any one of claims 13-15, wherein the LNP is a spleen-targeting LNP, and wherein the LNP has one or more of the following features: (i) in the PIL comprised by the LNP, n is 2 and A1and A2are alkyl chains having a carbon atom number of 16-18; (ii) in the PIL comprised by the LNP, A1and A2are amide bond-containing alkyl chains having a carbon atom number of 12, and n is 3-5; (iii) the PIL comprised by the LNP has a single free amino group or a free carboxyl group at the terminus; (iv) the PIL comprised by the LNP has a basic amino acid modification, preferably the PIL has a basic amino acid modification and an acetylation modification at the N-terminus; (v) the PIL comprised by the LNP has a proline modification, preferably the PIL has a proline modification and a free amino group at the N-terminus; (vi) the PIL comprised by the LNP has a tryptophan modification, preferably the PIL has a tryptophan modification and an acetylation modification at the N-terminus; or (vii) the PIL comprised by the LNP has an acidic amino acid modification, preferably the PIL has an acidic amino acid modification and a free carboxyl group at the C-terminus, preferably wherein the number of acidic amino acid modifications is less than or equal to 5, less than or equal to 4, or less than or equal to 3.

18. The LNP of any one of claims 13-15, wherein the LNP is a liver-targeting LNP, and wherein the LNP has one or more of the following features: (i) in the PIL comprised by the LNP, n is 4-5, or n is 3 and A1and A2are alkyl chains having a carbon atom number of 10-14; (ii) in the PIL comprised by the LNP, A1and A2are saturated alkyl chains or hydroxyl-containing alkyl chains having a carbon atom number of 12, and n is 3-5; or (iii) the PIL comprised by the LNP has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof, preferably the PIL has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine, or a combination thereof and an acetylation modification at the N-terminus.

19. The LNP of any one of claims 13-15, wherein the LNP is a lung-targeting LNP, and wherein the PIL comprised by the LNP has a basic amino acid modification, preferably the PIL has a basic amino acid modification and a free amino group at the N-terminus.

20. The LNP of any one of claims 13-15, wherein the LNP is a bone-targeting LNP, and wherein the PIL comprised by the LNP has a phosphorylation modification, for example the PIL has a phosphorylation modification at the N-terminus or the C-terminus, preferably a bisphosphonate modification, more preferably an alendronic acid modification.

21. The LNP of any one of claims 13-15, wherein the LNP is a liver- and spleen- dual targeting LNP, wherein in the PIL comprised by the LNP, A1and A2are ester bond- containing alkyl chains having a carbon atom number of 12. ​ ​ 22. The LNP of any one of claims 13-21, wherein the active agent is a chemically modified or unmodified nucleic acid molecule, such as a DNA molecule or an RNA molecule, preferably the mass ratio of total lipids to active agent in the LNP is about 1-100:

1.

23. A pharmaceutical composition comprising the LNP of any one of claims 13-22 and a pharmaceutically acceptable carrier.

24. A method of delivering an active agent to a target organ or target cell or preventing or treating a disease, the method comprising administering to a subject in need thereof or an ex vivo cell or tissue an effective amount of the LNP of any one of claims 13-22 or the pharmaceutical composition of claim 23.

25. Use of the LNP of any one of claims 13-22 or the pharmaceutical composition of claim 23 in the manufacture of a medicament for treating or preventing a disease.

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