Thymus targeting peptidyl ionizable lipid and nanoparticles thereof

Abstract

The invention relates to thymus targeting peptidyl ionizable lipids and nanoparticles thereof. More specifically, the present invention relates to peptidyl ionizable lipids, thymus targeted lipid nanoparticles comprising the same, and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of biology, and more particularly to peptide-based ionizable lipids, thymus-targeted lipid nanoparticles comprising peptide-based ionizable lipids, and their uses. Background Art

[0002] By enabling ribonucleic acid (messenger RNA, mRNA) to encode specific antigen fragments, gene editing proteins or other functional proteins, it can be widely applied to the development of infectious disease vaccines, cancer vaccines, gene therapies, and tissue repair. Due to its large size, high negative charge, and susceptibility to enzymatic degradation, the in vivo delivery and intracellular translation and expression of mRNA highly depend on delivery vectors. Among them, lipid nanoparticles (LNPs), as one of the most advanced mRNA delivery vectors currently, have been widely used in the research of novel mRNA-LNP-based clinical and preclinical gene therapies due to their excellent properties of high efficiency and safety in delivering mRNA in vivo, and have accelerated the successful approval of two COVID-19 mRNA vaccines (Comirnaty and Spikevax), making outstanding contributions to the global fight against the novel coronavirus pandemic.

[0003] LNPs are usually obtained by mixing four components, namely ionizable lipids, phospholipids, cholesterol, and polyethylene glycol lipids, in a certain ratio and assembling them with mRNA. They are relatively mature in the development of liver-targeted delivery and local intramuscular injection-related therapies. However, the mRNA targeted delivery to other extrahepatic organs is still in its infancy, which also limits the wide application of the mRNA-LNP technology in the treatment of diseases related to extrahepatic lesions. Currently, the main strategies for achieving extrahepatic mRNA targeted delivery include: (1) optimization of the LNP formulation; (2) development of novel targeted ionizable lipids; (3) modification of active targeting ligands; and (4) selection of the administration route, etc. Among them, although the optimization of the LNP formulation can achieve extrahepatic mRNA targeted delivery, its target organs are still mainly limited to the lungs and spleen; although the modification of active targeting ligands breaks through the limitation that mRNA is mainly expressed in the liver, spleen, and lungs, its preparation process is relatively cumbersome and it is difficult to produce on a large scale. Compared with the former two, the development of novel targeted ionizable lipids can avoid the disadvantages of the above strategies to a certain extent and has great application potential. Through rational lipid structure design, researchers have developed ionizable lipids that can target organs such as the liver, spleen, lungs, lymph nodes, and bone marrow. However, there is still no report on ionizable lipids that can target the thymus. In addition, the thymus is an important central immune organ in humans, where some immune cells grow, mature, and differentiate, playing an important role in the body's immune system. Therefore, the development of ionizable lipids and their nanoparticles with thymus targeting ability is of great significance for the development of related therapies for thymic lesions. Summary of the Invention

[0004] Based on the above research background in this field, in this study, the inventors proposed a design scheme for thymus-targeted peptidyl ionizable lipids, synthesized novel peptidyl ionizable lipids that can deliver mRNA to the thymus for expression, and obtained mRNA-LNPs that can highly specifically deliver mRNA to the thymus for expression by designing mRNA containing miR-126 binding sites (126ts mRNA).

[0005] In a first aspect, the present invention relates to a peptidyl ionizable lipid of formula I,

[0006]

[0007] wherein,

[0008] o is an integer from 1 to 30,

[0009] m and n are integers from 0 to 30,

[0010] p is an integer from 0 to 10,

[0011] X is independently O or S each time it appears,

[0012] A1 and A3 are each independently a hydrogen atom or an alkyl group each time they appear.

[0013] R2 and R4 each independently represent the side chains contained in the amino acid building blocks of the ionizable lipid, where R2 is a natural amino acid side chain or a non-natural amino acid side chain, and where R4 is any amino acid side chain with a pKa greater than or equal to about 6.0.

[0014] A4 and A5 are hydrophobic tails and are each independently an optionally substituted saturated or unsaturated, linear or branched alkyl chain having 4 to 25 carbon atoms, where the alkyl chain optionally contains one or more linking groups L selected from amide bonds, ester bonds, disulfide bonds, ketal bonds, ether bonds, or combinations thereof.

[0015] R6 represents the C-terminus of the peptidyl ionizable lipid, and R6 is a hydroxyl group or is modified with an amino group, an amino acid, and / or other functional groups.

[0016] In certain embodiments, R4 contains a basic group capable of accepting a proton, such as an amino group, an imidazole group, or a guanidine group. In certain embodiments, R4 is any amino acid side chain with a pKa greater than or equal to about 5.5, greater than or equal to about 6.0, greater than or equal to about 6.5, greater than or equal to about 7.0, greater than or equal to about 7.5, or greater than or equal to about 8.0, greater than or equal to about 8.5, greater than or equal to about 9.0, greater than or equal to about 9.5, greater than or equal to about 10.0, greater than or equal to about 10.5. In certain embodiments, R4 is any amino acid side chain with a pKa less than or equal to about 14.0, less than or equal to about 13.5, less than or equal to about 13.0, less than or equal to about 12.5, less than or equal to about 12.0.

[0017] In certain embodiments, R4 is a basic natural amino acid side chain such as a lysine side chain (-CH 2 CH 2 CH 2 CH 2 NH 2 ), an arginine side chain (-CH 2 CH 2 CH 2 NHC(NH)NH 2 ), or a histidine side chain (-CH 2 C 3 H 3 N 2 ).

[0018] In some embodiments, R4 is a side group of a basic non-natural amino acid. In some embodiments, the basic non-natural amino acid can be an amino acid or a combination thereof in which a basic natural amino acid is substituted with a substituent such as an alkyl, aryl, seleno, halogen (such as fluorine, chlorine, bromine or iodine), hydroxyl, nitro, phosphonic acid or amino group, and the substitution preferably does not change the positive charge of the side group of the basic natural amino acid. In some embodiments, the basic non-natural amino acid is selected from lysine derivatives such as 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn) and homolysine (hLys); arginine derivatives such as 2-amino-3-guanidinopropionic acid (Gpr), 2-amino-4-guanidinobutyric acid (Gbt) and homoarginine (hArg); histidine derivatives such as 1-methyl-histidine (1-MeHis), 3-methyl-histidine (3-MeHis) and homohistidine (hHis).

[0019] In certain embodiments, R4 has the formula -C 1-10 Alkyl-NH 2 , Formula-C 1-10 Alkyl-NHC(NH)NH 2 Or-C 1-10 Alkyl-C 3 H 3 N 2 , where C 1-10 Alkyl is a linear, branched, or cyclic optionally substituted alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. In certain embodiments, the optionally substituted alkyl group is an alkyl group substituted with a substituent such as an alkyl group, an aryl group, a seleno group, a halogen group (e.g., a fluorine group, a chlorine group, a bromine group, or an iodine group), a hydroxyl group, a nitro group, a phosphonic acid group, or an amino group.

[0020] In certain embodiments, the peptidyl ionizable lipid has the following Formula II,

[0021] ,

[0022] wherein o, m, n, p, X, A1, A3, R2, A4, A5 and R6 are as defined in Formula I.

[0023] In certain embodiments, R2 is a natural amino acid side group, and the natural amino acid is preferably selected from serine, threonine, histidine, lysine, arginine, tyrosine, tryptophan and phenylalanine, and more preferably selected from serine, histidine and tyrosine. In certain embodiments, R2 is a serine side group (-CH 2 OH), histidine side groups (-CH 2 C 3 H 3 N 2 ) or the tyrosine side group (-CH2 C 6 H 4 OH).

[0024] In certain embodiments, R2 is a non-natural amino acid side chain group. In certain embodiments, the non-natural amino acid can be an amino acid in which a natural amino acid is substituted by a substituent such as an alkyl group, an aryl group, a seleno group, a halogen (e.g., fluorine, chlorine, bromine or iodine), a hydroxyl group, a nitro group, a phosphonic acid or an amino group, or a combination thereof. In certain embodiments, R2 is a non-natural amino acid side chain group, and the non-natural amino acid is selected from serine derivatives such as homoserine (Hse), phosphoserine (pSer), selenocysteine (Sec); histidine derivatives such as 1-methyl-histidine (1-MeHis), 3-methyl-histidine (3-MeHis) and homohistidine (hHis); and tyrosine derivatives such as p-hydroxyphenylalanine (pHpa), 3-nitro-tyrosine (3-NT) and 3,5-diiodotyrosine (DIT).

[0025] In certain embodiments, R2 has the formula -C 1-10 alkyl-C 6 H 4 OH, wherein C 1-10 alkyl is a linear, branched or cyclic optionally substituted alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. In certain embodiments, the optionally substituted alkyl group is an alkyl group substituted by a substituent such as an alkyl group, an aryl group, a seleno group, a halogen (e.g., fluorine, chlorine, bromine or iodine), a hydroxyl group, a nitro group, a phosphonic acid or an amino group. In certain embodiments, one or more positions on the benzene ring in the formula -C 1-10 alkyl-C 6 H 4 OH are substituted by substituents such as an alkyl group, an aryl group, a seleno group, a halogen (e.g., fluorine, chlorine, bromine or iodine), a hydroxyl group, a nitro group, a phosphonic acid or an amino group.

[0026] In certain embodiments, p is an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, p is 1, that is, the corresponding amino acid building block is an amino acid building block in which the primary amino group of the side chain of 2,3-diaminopropionic acid (Dap) is alkylated. In certain embodiments, p is 2, that is, the corresponding amino acid building block is an amino acid building block in which the primary amino group of the side chain of 2,4-diaminobutyric acid (Dab) is alkylated. In certain embodiments, p is 3, that is, the corresponding amino acid building block is an amino acid building block in which the primary amino group of the side chain of ornithine is alkylated. In certain embodiments, p is 4, that is, the corresponding amino acid building block is an amino acid building block in which the primary amino group of the side chain of lysine is alkylated. In certain embodiments, p is preferably 1, 2, 3 or 4, more preferably 2.

[0027] In certain embodiments, A1 and A3 are each independently a hydrogen atom or a C 1-6 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl or cyclohexyl.

[0028] In certain embodiments, the number of carbon atoms of A4 or A5 is 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 A4 and A5 contain one or more linking groups L, the number of carbon atoms of the alkyl chain refers to the number of carbon atoms contained in the hydrocarbon group connected after the last linking group. In certain embodiments, when A4 and A5 are an alkyl chain containing an amide bond or an alkyl chain containing an ester bond, the number of carbon atoms refers to the number of carbon atoms contained in the hydrocarbon group connected after the amide bond or the ester bond.

[0029] In certain preferred embodiments, the alkyl chain of A4 or A5 of the present invention contains 0, 1, 2, 3, 4, 5, or 6 linking groups L. In certain preferred embodiments, the linking group L may be a biodegradable group, such as an ester bond that is stable at physiological pH but hydrolyzed by enzymes in tissues and cells, a disulfide bond sensitive to the reducing intracellular environment, a ketothiol bond responsive to ROS, etc.

[0030] In certain embodiments, A4 and A5 are each independently -(B1-L) s -B2, where s is an integer from 0 to 6, such as 0, 1, 2, 3, 4, 5 or 6, where B1 is independently absent or is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms in each (B1-L) unit, L is independently absent or is a linking group selected from an amide bond, an ester bond, a disulfide bond, a ketothiol bond, an ether bond or a combination thereof in each (B1-L) unit, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms.

[0031] In certain embodiments, B1 is absent in the (B1-L) unit, i.e., the (B1-L) unit is -L-. In certain embodiments, B1 is each independently an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, preferably B1 is an optionally substituted 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. In certain embodiments, B1 is substituted with a hydroxyl group.

[0032] 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 ketal bond, an ether bond, or a combination thereof.

[0033] In certain embodiments, B2 is an optionally substituted saturated or unsaturated hydrocarbyl group having 1 to 25 carbon atoms, preferably B2 is an optionally substituted 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. In certain embodiments, B2 is hydroxy-substituted.

[0034] In certain embodiments, A4 and A5 are, 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, where each B1 is absent or independently is an optionally substituted saturated or unsaturated hydrocarbyl group having 1 to 25 carbon atoms, where L is absent or independently is a linking group selected from an amide bond, an ester bond, a disulfide bond, a ketal bond, an ether bond, or a combination thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbyl group having 1 to 25 carbon atoms.

[0035] In certain embodiments, A4 and A5 are, independently of each other, selected from: a saturated alkyl chain, an unsaturated alkyl chain, a hydroxyalkyl chain, an amide bond-containing alkyl chain, an ester bond-containing alkyl chain, a disulfide bond-containing alkyl chain, a ketal bond-containing alkyl chain, a branched alkyl chain.

[0036] In certain embodiments, A4 and A5 are, independently of each other, selected from: -CH 2 CH 2 (CH 2 ) q CH 2 CH 3 、-CH 2 CHOH(CH 2 ) q CH 2 CH 3 、-CH 2 CH 2 CONHCH 2 CH 2 (CH 2 ) q CH 2 CH 3 、-CH 2 CH 2 NHCOCH 2 CH2 (CH 2 ) q CH 2 CH 3 、 -CH 2 CH 2 COOCH 2 CH 2 (CH 2 ) q CH 2 CH 3 、 -CH 2 CH 2 OOCCH 2 CH 2 (CH 2 ) q CH 2 CH 3 where q is an integer from 0 to 21, such as 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.

[0037] In certain embodiments, A4 and A5 are the same. In certain embodiments, A4 and A5 are each independently a hydroxyalkyl chain, preferably -CH 2 CHOH(CH 2 ) q CH 2 CH 3 where q is an integer from 0 to 21, such as 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.

[0038] In certain embodiments, o is an integer from 1 - 30, m and n are integers from 0 - 30, such as o, m and n are each independently 0, 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. In certain embodiments, o is preferably 1, 2, 3, 4 or 5. In certain embodiments, o is preferably an integer greater than 3, such as 3, 4, 5 or 6.

[0039] In certain embodiments, the PIL has a free carboxyl group at the C - terminus (R6 represents -OH) or has an amidation modification (R6 represents -NH 2 ). In certain embodiments, the PIL has both an amidation modification and an amino acid modification at the C - terminus (R6 represents -(aa)x - NH 2or has an amino acid modification and a free carboxyl group (R6 represents -(aa)x), where (aa)x is one or more amino acid residues.

[0040] In certain embodiments, the amino acid modifying the C-terminus of the PIL is a natural amino acid or a non-natural amino acid. In certain embodiments, the amino acid modifying 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).

[0041] In certain embodiments, the PIL has 1 to 10 amino acid modifications at the C-terminus, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0042] In certain embodiments, the peptidyl ionizable lipid is selected from:

[0043] , ,

[0044] , ,

[0045] , ,

[0046] , ,

[0047] , ,

[0048] .

[0049] In certain embodiments, the peptidyl ionizable lipid is a thymus-targeted peptidyl ionizable lipid. In certain embodiments, a thymus-targeted peptidyl ionizable lipid means that when a lipid nanoparticle comprising the thymus-targeted peptidyl ionizable lipid is delivered in vivo, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 96% of the lipid nanoparticles are expressed in the thymus.

[0050] In a second aspect, the present invention relates to a thymus-targeted lipid nanoparticle (LNP) comprising the peptidyl ionizable lipid of the first aspect, an optional co-lipid, and an optional active agent. In certain embodiments, the co-lipid includes phospholipids, steroids, and / or PEG lipids.

[0051] In certain embodiments, based on the total lipids, the lipid nanoparticles comprise from about 10 mol% to about 100 mol% of the peptidyl ionizable lipid, from about 0 mol% to about 30 mol% of phospholipids, from about 0 mol% to about 70 mol% of steroids, and / or from about 0 mol% to about 10 mol% of PEG lipids.

[0052] In certain embodiments, the active agent is a polynucleotide containing one or more regulatory sequences that reduce the expression of the polynucleotide in non-thymic organs.

[0053] In certain embodiments, the non-thymic organs are selected from: lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, brain stem, cerebellum, spinal cord, eye, ear, tongue, or skin. In certain embodiments, the non-thymic organ is the lung.

[0054] In certain embodiments, the one or more regulatory sequences include miRNA binding sites. In certain embodiments, the miRNA binding sites bind miRNAs selected from the group consisting of miR-122, miR-133, miR-206, miR-208, miR-17-92, miR-126, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, miR-204, miR142, miR150, or combinations thereof. In certain embodiments, the miRNA binding sites bind let-7, miR-133, miR-126, or combinations thereof. The miRNA binding site comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99% identity to the sequence shown in SEQ ID NO: 1.

[0055] In certain embodiments, 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 lipid to active agent in the LNP is about 1-100:1.

[0056] In certain embodiments, the active agent of the present invention 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, optionally selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA probes, DNA aptamers, DNA nanoframes, DNA primers, homology repair DNA templates, immunostimulatory DNA, or combinations thereof.

[0057] In certain embodiments, the active agent of the present invention 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 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 nanoscaffold, RNA ribonucleoprotein complex or a combination thereof. In certain embodiments, the active agent of the present invention is a chemically modified messenger RNA (mRNA), preferably any mRNA encoding a gene editing element.

[0058] In certain embodiments, the active agent of the present invention can be a combination of the above DNA molecules and RNA molecules. In certain embodiments, the active agent is a gene therapy agent. In certain embodiments, the mass ratio of total lipid to active agent in the LNP is about 1 - 100:1.

[0059] In a third aspect, the present invention provides a pharmaceutical composition comprising the LNP according to the second aspect and a pharmaceutically acceptable carrier.

[0060] In a fourth aspect, the present invention provides a method for delivering an active agent to the thymus or thymocytes or for preventing or treating a disease, the method comprising administering an effective amount of the LNP according to the second aspect or the pharmaceutical composition according to the third aspect to a subject in need or to isolated cells or tissues.

[0061] The present invention also provides the use of the LNP according to the second aspect or the pharmaceutical composition according to the third aspect in the preparation of a drug for treating or preventing a disease.

[0062] The present invention also provides a drug for treating or preventing a disease, the drug comprising the LNP according to the second aspect or the pharmaceutical composition according to the third aspect.

[0063] The present invention also provides the LNP according to the second aspect or the pharmaceutical composition according to the third aspect for treating or preventing a disease.

[0064] In certain embodiments, the disease is any disease for which the active agent of the present invention 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 inducing an immune response. In certain embodiments, the disease is cancer.

[0065] In certain embodiments, the disease is a disease that can be improved by targeted delivery of an active agent to the thymus. In certain embodiments, the disease is a disease that can be improved by enhancing the immune function in the thymus, such as cancer treated by in vivo generation of CAR-T cells. In certain embodiments, the disease is selected from cancer, thymic hyperplasia, thymic tumors, autoimmune diseases, organ transplant rejection, thymic atrophy such as age-related thymic atrophy, or a combination thereof.

[0066] In a fifth aspect, the present invention also provides the use of the peptidyl ionizable lipid of the first aspect in the preparation of an LNP targeting the thymus.

[0067] In summary, this study synthesized a series of PIL lipids composed of artificial alkylated amino acid AIFA and natural amino acids. Through a screening method of in vivo delivery of Luc mRNA, PILs and corresponding LNPs capable of highly specifically delivering mRNA to the thymus for expression were discovered. By introducing miRNA binding sites, their expression in other organs outside the thymus was reduced, further enhancing the targeting specificity of thymus-targeting LNPs. The obtained mRNA-LNPs have certain potential for developing mRNA therapies for treating diseases related to thymic lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 . Nα-Fmoc protected amino acid molecular building blocks with alkylated primary amino groups in the side chain.

[0069] Figure 2 . Representative chemical structures and 1H NMR spectra of Nα-Fmoc protected alkylated amino acids.

[0070] Figure 3 . Chemical structures of the synthesized PIL lipids.

[0071] Figure 4 . Chemical structures of the synthesized PIL lipids.

[0072] Figure 5 . Representative PIL lipid Am-KYa12K4 structure and 1H NMR spectrum.

[0073] Figure 6. Encapsulation efficiency of different PIL lipids for Luc mRNA.

[0074] Figure 7 . Hydrodynamic diameter of LNP prepared from different PIL lipids.

[0075] Figure 8 . Surface potential of LNP prepared from different PIL lipids.

[0076] Figure 9 . Polydispersity index of LNP prepared from different PIL lipids.

[0077] Figure 10 . Bioluminescence imaging of major organs in mice mediated by different Luc mRNA-LNP.

[0078] Figure 11 . Fluorescence intensity comparison of thymus mediated by different PIL Luc mRNA-LNP

[0079] Figure 12 . Expression percentage of a12Dab4 and Am-KYa12K4 mRNA-LNP in each major organ.

[0080] Figure 13 . Thymus-targeted Am-KYa12K4 LNP encapsulating miR126ts-Luc mRNA to reduce mRNA expression in the lung.

[0081] Figure 14 . In vivo delivery efficiency of thymus-targeted Am-KYa12K4 LNP encapsulating miR126ts-Luc mRNA and thymus mRNA expression percentage. Detailed implementation manners

[0082] Definition

[0083] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0084] In the present invention, the singular forms "a", "an" and "the" are intended to include the plural forms. In the present invention, the term "and / or" includes any and all combinations of one or more of the related listed items. It should also be understood that when the terms "comprises" or "comprising" are used in this specification, they specify the presence of the 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 should also be understood that when using the terms "comprises" or "comprising", they cover the meanings of "consisting essentially of..." or "consisting of...". The term "or combinations thereof" refers to a combination including at least one of the foregoing elements.

[0085] Unless otherwise indicated, all numbers used in this specification and the claims should be understood to be modified in all instances by the term "about." As used in the present invention, the term "about" or "approximately," when applied to one or more values of interest, refers to a value that is close to the stated reference value or within an acceptable error range of the specific value. In one case, the term "about" refers to any value within a variation range of up to ±10% of the value modified by the term "about," including both integer and fractional components. Alternatively, in accordance with practices in the art, "about" may mean within 3 or more standard deviations, within 5-fold, or within 2-fold.

[0086] As used herein, the term "alkyl chain" includes saturated alkyl chains and unsaturated alkyl chains, and also includes linear, branched, or cyclic alkyl chains. In the definitions of the present invention, the "alkyl chain" may contain one or more linking groups L in addition to the carbon skeleton. The term "unsaturated alkyl chain" refers to an alkyl chain containing one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unsaturated bonds such as carbon-carbon double bonds or carbon-carbon triple bonds. The term "alkyl chain" as used herein also includes optionally substituted alkyl chains, such as hydroxy-substituted alkyl chains.

[0087] As used herein, the term "hydrocarbyl" refers to a group formed by removing one or more hydrogen atoms from a hydrocarbon molecule (hydrocarbon). In certain embodiments, the "hydrocarbyl" of the present invention refers to an aliphatic hydrocarbyl. In certain embodiments, the "hydrocarbyl" of the present invention refers to a linear or branched alkyl, alkenyl, or alkynyl. For example, the "saturated or unsaturated hydrocarbyl having 1 to 25 carbon atoms" of the present invention refers to a linear or branched alkyl, alkenyl, or alkynyl 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 "alkyls" include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Representative "alkenyls" include vinyl, 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 "alkynyls" include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0088] As used herein, the term "lower alkyl" refers to an alkyl containing 10 or fewer, preferably 6 or fewer carbon atoms, such as 1, 2, or 3 carbon atoms.

[0089] As used herein, the terms "branched" or "branching" mean that a given structure such as an alkyl chain, a hydrocarbon group, or an alkyl group contains a tertiary carbon atom bonded to three other carbon atoms or a quaternary carbon atom bonded to four other carbon atoms.

[0090] As used herein, the term "optionally substituted" means that 1 to 6 hydrogen groups in a given structure are replaced by groups of specific substituents, and the specific substituents include, but are not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro. Preferably, "optionally substituted" means "optionally hydroxyl-substituted". Preferably, 0, 1, 2, or 3 hydrogen groups in the alkyl chain are replaced by hydroxyl groups.

[0091] In the present invention, the symbol "-" means a single bond, does not indicate any preferred stereochemistry, and encompasses all stereoisomers and mixtures thereof.

[0092] In the present invention, the term "containing one or more linking groups L" means that an alkyl chain 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", where the linking group L or L1 and L2 are independently selected from an amide bond, an ester bond, a disulfide bond, a ketal bond, an ether bond, or a combination thereof. In certain embodiments, the alkyl chain of A4 or A5 in the present invention does not contain a linking group or contains 1, 2, 3, or 4 linking groups.

[0093] In the present invention, the term "saturated alkyl chain" or "alkyl chain a" means a linear or branched alkyl having 4 to 25 carbon atoms and having no unsaturated bonds. In certain embodiments, the saturated alkyl chain of the present invention is an alkyl that is not substituted by any substituents. In certain embodiments, the saturated alkyl chain of the present invention is -CH 2 CH 2 (CH 2 ) q CH 2 CH 3 , where q is an integer from 0 to 21.

[0094] In the present invention, the term "unsaturated alkyl chain" means a linear or branched hydrocarbon group having 4 to 25 carbon atoms and containing one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, unsaturated bonds such as carbon-carbon double bonds or carbon-carbon triple bonds. Unsaturated alkyl chains that can be used as A4 or A5 in the present invention are known in the art, for example, see R1 and R2 in WO2010054406A1.

[0095] In the present invention, the term "hydroxyalkyl chain" or "e-alkyl chain" refers to a linear or branched hydrocarbon group having 4 to 25 carbon atoms and substituted with at least one hydroxyl group. In certain embodiments, the hydroxyalkyl chain of the present invention is a linear or branched alkyl group having 4 to 25 carbon atoms and substituted with at least one hydroxyl group, such as 1, 2, 3, 4, 5 or 6 hydroxyl groups. In certain embodiments, the hydroxyalkyl chain of the present invention is -CH 2 CHOH(CH 2 ) q CH 2 CH 3 , where q is an integer from 0 to 21.

[0096] The alkyl chain of A4 or A5 of the present invention optionally further includes one or more linking groups L, and the linking groups L are independently selected from an amide bond, an ester bond, a disulfide bond, a ketal bond, an ether bond or a combination thereof.

[0097] In certain preferred embodiments, the alkyl chain of A4 or A5 of the present invention contains 0, 1, 2, 3 or 4 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 hydrolyzed by enzymes in tissues and cells, a disulfide bond sensitive to the reducing intracellular environment, a ketal bond responsive to ROS, etc. The effects of adding biodegradable groups to the alkyl chain are known in the art, for example, see WO2011153493A2, WO2013086354A1.

[0098] In the present invention, the term "amide bond-containing alkyl chain" or "aam alkyl chain" refers to an alkyl chain containing at least one amide bond (-C(=O)-NH- or -NH-C(=O)-). In the present invention, when A4 or A5 is an amide bond-containing alkyl chain, the number of carbon atoms of A4 or A5 refers to the number of carbon atoms of the hydrocarbon group connected after the amide bond. In certain embodiments, the amide bond-containing alkyl chain of the present invention is -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, where each B1 is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, where L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a ketal bond, an ether bond or a combination thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms. In certain embodiments, the amide bond-containing alkyl chain of the present invention is -CH 2 CH 2 CONHCH 2 CH 2 (CH 2 ) q CH 2 CH 3 or -CH2 CH 2 NHCOCH 2 CH 2 (CH 2 ) q CH 2 CH 3 , where q is an integer from 0 to 21.

[0099] In the present invention, the term "alkyl chain containing an ester bond" or "aat alkyl chain" refers to an alkyl chain containing at least one ester bond (-C(=O)-O- or -O-C(=O)-). In the present invention, when A4 or A5 is an alkyl chain containing an ester bond, the number of carbon atoms of A4 or A5 refers to the number of carbon atoms of the hydrocarbon group connected after the ester bond. In certain embodiments, the alkyl chain containing an ester bond of the present invention is -B1-COO-B1-L-B2 or -B1-OOC-B1-L-B2, where each B1 is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group with 1 to 25 carbon atoms, where L is absent or independently a linking group selected from an amide bond, an ester bond, a disulfide bond, a ketal dithiol bond, an ether bond, or a combination thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group with 1 to 25 carbon atoms. In certain embodiments, the alkyl chain containing an ester bond of the present invention refers to -CH 2 CH 2 COOCH 2 CH 2 (CH 2 ) q CH 2 CH 3 or -CH 2 CH 2 OOCCH 2 CH 2 (CH 2 ) q CH 2 CH 3 , where q is an integer from 0 to 21.

[0100] In the present invention, the term "alkyl chain containing a disulfide bond" refers to an alkyl chain containing at least one disulfide bond (-S-S-), that is, at least one L in -(B1-L) s -B2 is a disulfide bond. The term "alkyl chain containing a ketal dithiol bond" refers to an alkyl chain containing at least one ketal dithiol bond (-S-C(CH 3 ) 2 -S-), that is, at least one L in -(B1-L) s -B2 is a ketal dithiol bond. The term "branched alkyl chain" refers to an alkyl chain containing at least one branched hydrocarbon group, for example, at least one B1 or B2 in -(B1-L) s -B2 is a branched hydrocarbon group.

[0101] Peptidyl ionizable lipid

[0102] In the present invention, the term "peptide-based ionizable lipid" or "PIL" refers to a peptide-based ionizable lipid composed of alkylated amino acid building blocks of the present invention and optionally natural or other non-natural amino acid building blocks connected by amide bonds.

[0103] In the present invention, the term "amino acid" includes D-amino acids and L-amino acids. In the present invention, the term "natural amino acid" refers to amino acids that naturally exist in organisms, including 20 different amino acids commonly found in proteins, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0104] In the present invention, the terms "unnatural amino acid", "non-canonical amino acid", and "artificial amino acid" refer to amino acids other than the above 20 common amino acids and not encoded by the genetic code in organisms, including alanine derivatives, arginine derivatives, asparagine derivatives, aspartic acid derivatives, cysteine derivatives, glutamine derivatives, glutamic acid derivatives, glycine derivatives, histidine derivatives, isoleucine derivatives, leucine derivatives, lysine derivatives, methionine derivatives, phenylalanine derivatives, proline derivatives, serine derivatives, threonine derivatives, tryptophan derivatives, tyrosine derivatives, and valine derivatives, etc. In the present invention, unnatural amino acids can be amino acids in which natural amino acids are substituted by substituents such as alkyl, aryl, seleno, halogen (such as fluorine, chlorine, bromine, or iodine), hydroxyl, nitro, phosphonic acid, or amino, or combinations thereof. In the present invention, unnatural amino acids include basic unnatural amino acids such as lysine derivatives such as 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine (Orn), and homolysine (hLys); arginine derivatives such as 2-amino-3-guanidinopropionic acid (Gpr), 2-amino-4-guanidinobutyric acid (Gbt), and homoarginine (hArg); histidine derivatives such as 1-methyl-histidine (1-MeHis), 3-methyl-histidine (3-MeHis), and homohistidine (hHis). In the present invention, unnatural amino acids also include serine derivatives such as homoserine (Hse), phosphoserine (pSer), selenocysteine (Sec); and tyrosine derivatives such as p-hydroxyphenylalanine (pHpa), 3-nitro-tyrosine (3-NT), and 3,5-diiodotyrosine (DIT).

[0105] In addition, non-natural amino acids that can be used in the present invention also include, for example, N-ethyl-β-alanine (Neb), sarcosine (Sar), α,β-diaminopropionic acid (Adp), β-amino-N-butyric acid (aBut or Bab), β-aminoisobutyric acid (Bai), α-aminoisobutyric acid (Aib), γ-aminobutyric acid (Gab), α-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), pipecolic acid (Pip), ornithine (Orn), tert-leucine (Tle), alloisoleucine (Ali), norleucine (Nle), 2-aminoheptanoic acid (Ahe), citrulline (Cit).

[0106] In the present invention, the term "basic amino acid" refers to an amino acid that has a net positive charge at neutral pH, such as lysine, arginine, and histidine.

[0107] The term "acidic amino acid" refers to an amino acid that has a net negative charge at neutral pH, such as glutamic acid and aspartic acid.

[0108] The term "pKa" refers to the negative logarithm of the acid dissociation constant and is used to quantify the ability of a substance (such as an amino acid or the side chain of an amino acid) to release protons in solution.

[0109] Lipid nanoparticle

[0110] In the present invention, the term "lipid nanoparticle" refers to a particle having a nanoscale size (e.g., 1-1,000 nm) that contains one or more lipids.

[0111] In certain embodiments, the lipid nanoparticles of the present invention comprise the peptide-based ionizable lipid of the present invention, optional co-lipids, and optional active agents.

[0112] In certain embodiments, the lipid nanoparticles of the present invention may have an average particle diameter between about 50 nm and about 200 nm, such as between about 100 nm and about 120 nm or between about 140 nm and about 190 nm. In certain embodiments, the surface charge of the lipid nanoparticles of the present invention may be between about -20 mV and about +20 mV. In certain embodiments, the surface charge of the lipid nanoparticles of the present invention is between about 0 mV and about +20 mV, such as between about +1 mV and about +20 mV, between about +2 mV and about +18 mV, between about +3 mV and about +15 mV, or between about +4 mV and about +13 mV. In certain embodiments, the polydispersity index (PDI) value of the lipid nanoparticles of the present invention is between about 0 and about 0.3, such as between about 0.1 and about 0.25, such as between about 0.13 and about 0.23.

[0113] In the present invention, the term "ionizable lipid" or "cationic lipid" refers to a lipid molecule that is protonated in an acidic environment (e.g., at a pH in the range of about 4.0 to 6.0) and remains neutral or weakly charged at physiological pH (e.g., about 7.4). In certain embodiments, in addition to the peptidyl ionizable lipids of the present invention, the lipid nanoparticles of the present invention optionally further comprise additional ionizable lipids, such as (9-heptadecanyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), [4-hydroxybutylazanediyl]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)-trimethylammonium (DOTAP), and the like.

[0114] The peptidyl ionizable lipid or peptidyl ionizable lipids may be included in the lipid nanoparticles in different amounts. For example, based on the total lipids, the lipid nanoparticles may include about 10 mol% to about 100 mol%, such as about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol% of ionizable lipid or peptidyl ionizable lipid.

[0115] In certain embodiments, the "active agent" as described in the present invention refers to any active substance intended to be delivered by lipid nanoparticles, such as therapeutic agents, immunomodulatory agents, etc. In certain embodiments, the active agent of the present invention is a nucleic acid molecule, such as antisense oligonucleotides (ASO), mRNA, siRNA, guide RNA (gRNA), viral vectors, 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 PEmax mRNA and engineered prime editing guide RNA (epegRNA). In certain embodiments, the mass ratio of total lipid to active agent in the LNP is about 1-100:1, such as about 20-80:1, such as about 40:1. In certain embodiments, the content of the active agent in the LNP is about 1 ng to about 10 μg, such as about 400 ng to 1 μg, about 500 ng, or about 600 ng.

[0116] In the present invention, the term "helper lipid" refers to a lipid in the lipid nanoparticle that contributes to its stability and delivery efficiency in addition to the cationic lipid. Examples of helper lipids in the present invention include, but are not limited to, phospholipids, steroids, PEG lipids, etc.

[0117] In the present invention, examples of phospholipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), etc. The phospholipids can be included in the lipid nanoparticles in different amounts. For example, based on the total lipid, the lipid nanoparticles can include about 0 mol% to about 30 mol%, such as about 5 mol% to about 30 mol%, about 8 mol% to about 15 mol% of phospholipids. In certain embodiments, the lipid nanoparticles can contain a molar ratio of phospholipid to 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.

[0118] In the present invention, examples of steroids include, but are not limited to, cholesterol and its derivatives, ergosterol, lanosterol, stigmasterol, sitosterol, etc. Among them, examples of cholesterol derivatives include, but are not limited to, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, etc. The steroids can be included in the lipid nanoparticles in different amounts. For example, based on the total lipids, the lipid nanoparticles can include about 0 mol% to about 70 mol%, such as about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol% of steroids. In certain embodiments, the lipid nanoparticles can have a molar ratio of steroids to peptide-based ionizable lipids 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 therefrom. In certain embodiments, the molar ratio is about 1:1 to about 6:1 such as 2:1 or 3:1.

[0119] In the present invention, PEG lipids refer to any complex of polyethylene glycol (PEG) and lipids, and examples thereof include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, etc. In certain embodiments, the PEG lipid is PEG-modified distearoyl phosphatidylethanolamine 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 molecular weight of the PEG modification is 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 nanoparticles in different amounts. For example, based on the total lipid, the lipid nanoparticles can include about 0 mol% to about 10 mol%, such as 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. In certain embodiments, the lipid nanoparticles can have a molar ratio of PEG lipid to peptide-based 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.

[0120] In certain embodiments, the LNP of the present invention is a thymus-targeting LNP. "Targeting" or "specificity" in the present invention means that when the LNP is delivered into a living body, the expression ratio in a specific organ or tissue exceeds about 50%, such as exceeds about 60%, exceeds about 65%, exceeds about 70%, exceeds about 75%, exceeds about 80%, and more preferably exceeds about 90%. Alternatively, "thymus targeting" in the present invention means that when administered in vivo, at least about 25%, at least about 50%, at least about 75%, at least about 80% or at least about 90% of the administered amount of the lipid nanoparticles is delivered to the thymus.

[0121] miRNA binding site

[0122] In the present invention, the terms "miRNA binding site", "microRNA binding site", "microRNA target site", or "miRts" are used interchangeably and refer to a sequence that has sufficient complementarity to all or a region of a miRNA to interact, associate, or bind with the miRNA.

[0123] In the present invention, the "miRNA binding site" can be located at any position of an active agent such as mRNA, for example, in the 5'UTR, between the 5'UTR and the CDS, between the 3'UTR and the CDS, or in the 3'UTR. In certain embodiments, the miRNA binding site of the present invention is capable of promoting the regulation, such as reduction, of the expression of an active agent such as mRNA mediated by miRNA.

[0124] In the present invention, the "miRNA binding site" encompasses the binding sites of any known miRNA expressed in non-thymic organs. Examples of tissues where miRNAs are known to regulate mRNA and thus protein expression include, but are not limited to, the liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), spleen (miR142), lymphoid cells (miR150), and lung epithelial cells (let-7, miR-133, miR-126), including the miRNAs disclosed in WO2024026257 or WO2017062513A1.

[0125] Example

[0126] First, Fmoc group-protected alkylated ionizable amino acid building blocks (AIFAs) were synthesized, including Fmoc-L-Lys(a12)-OH, Fmoc-L-Lys(e12)-OH, and Fmoc-L-Dab(a12)-OH ( Figure 1). Among them, the specific synthesis methods of Fmoc-L-Lys(a12)-OH and Fmoc-L-Dab(a12)-OH are as follows: Fmoc-L-lysine (Lys) and Fmoc-L-diaminobutyric acid (Dab) are respectively dissolved in 1,2-dichloroethane containing 1% acetic acid (by volume), and activated at room temperature for 1 hour; then sodium triacetoxyborohydride is added, and the reaction is carried out at room temperature for 3 days with continuous gentle stirring; the reaction solution is washed with 0.1% trifluoroacetic acid aqueous solution and concentrated, and purified by flash column chromatography to finally obtain a pale yellow viscous solid. The specific synthesis method of Fmoc-L-Lys(e12)-OH is as follows: Boc-L-lysine and dodecene oxide are dissolved in ethanol, and the reaction is carried out at 80 °C for 3 days with continuous gentle stirring; after purification by flash column chromatography, Boc-protected dodecylated lysine is obtained; the Boc-protected dodecylated lysine is dissolved in dichloromethane, and trifluoroacetic acid is added thereto, and the reaction solution is reacted at room temperature for 4 h to remove the Boc group; the dodecylated lysine after removing the Boc group is dissolved in a mixed solvent of acetonitrile / tetrahydrofuran containing N,N-diisopropylethylamine, and under ice bath conditions, a tetrahydrofuran solution of fluorenylmethoxycarbonyl succinimide is slowly added dropwise thereto. After the addition is completed, it is transferred to room temperature and reacted overnight; the reaction solution is post-treated and purified by flash column chromatography to obtain a pale yellow viscous solid. The proton nuclear magnetic resonance spectrum shows the correct synthesis of the final product. The chemical structure and NMR results of the representative final product are as Figure 2 shown.

[0127] Subsequently, based on the solid-phase peptide synthesis technology of the Fmoc strategy, a series of PIL lipids were synthesized using the above-synthesized alkylated amino acid building blocks and natural amino acid building blocks. Their names and specific chemical structures are as Figures 3 - 4 shown. Among them, "Am-" represents that the N-terminus of the PIL lipid is a primary amino group, "-Ca" represents that the C-terminus of the PIL lipid is a carboxyl group, a is a saturated alkyl chain obtained by aldehyde reductive amination, and e is a hydroxyl-containing alkyl chain obtained by epoxy ring opening; Lys / K is lysine, and Dab is 2,4-diaminobutyric acid. The proton nuclear magnetic resonance spectrum shows the correct synthesis of the final product. The chemical structure and NMR results of the representative final product Am-KYa12K4 are as Figure 5 shown.

[0128] mRNA-LNP was prepared based on the above-mentioned PIL. An ethanol solution of lipids was configured with a molar percentage of PIL: distearoyl phosphatidylcholine (DSPC): cholesterol (Chol): dimyristoyl polyethylene glycol (DMG-PEG) = 46.3:9.4:42.7:1.6. Using the microfluidic preparation technology, the ethanol solution of lipids was mixed with the sodium citrate solution (10 mM, pH 3.0) of Luc mRNA at a certain flow rate according to a mass ratio of total lipid / luciferase mRNA (Luc mRNA) = 40:1. After dialysis with phosphate (PBS) buffer, a series of PIL-based Luc mRNA-LNP were prepared, and their physicochemical properties were characterized.

[0129] First, the encapsulation efficiency of the above-mentioned PIL for Luc mRNA was determined. Based on the RiboGreen fluorescent dye, the encapsulation efficiency of PIL LNP for Luc mRNA was calculated by measuring the concentration of free mRNA in the mRNA-LNP solution and the total mRNA concentration in the solution after fully lysing the LNP with Triton X-100, respectively. The results showed that the encapsulation efficiency of PIL lipids for mRNA was between 75.9% and 98.8% ( Figure 6 ), indicating that the synthesized PIL had a high encapsulation efficiency for mRNA.

[0130] Next, the hydrodynamic diameter, surface charge and polydispersity index of PIL mRNA-LNP were measured using dynamic light scattering technology ( Figures 7 - 9 ). The test results showed that the hydrodynamic diameter of the above-mentioned PIL mRNA-LNP was between 140 nm and 190 nm, and the particle size distribution of the prepared LNP was relatively uniform, with a PDI value between 0.13 and 0.23. In terms of the surface charge of LNP, the inventors found that the surface charge of PIL without natural amino acids was nearly neutral; the surface charge of PIL lipid a12K4E-Ca containing glutamic acid was about -10.6 mV; while the LNP formed by PIL containing lysine carried a positive charge, and the surface potential was between +4 mV and +13 mV. Moreover, with the increase in the number of lysine residues, the positive charge on the surface of the nanoparticles became stronger.

[0131] Furthermore, the efficiency of PIL mRNA-LNP in delivering Luc mRNA (1.0 mg / kg mRNA) in vivo was investigated. Six hours after tail vein injection, the expression of mRNA in organs such as the liver, spleen, lung, heart, kidney and thymus was analyzed. Figure 10). The data results show that a12K4 mainly mediates the expression of mRNA in the liver, and also has a certain expression in the spleen and lungs, while almost no mRNA expression can be observed in the thymus; after modifying its N-terminus with lysine or arginine, it can be seen that the modified Am-Ka12K4, Am-K2a12K4, Am-K3a12K4 and Am-Ra12K4 significantly reduce the expression of mRNA in the liver and spleen, and a certain mRNA expression can be observed in the thymus; after modifying its C-terminus with glutamic acid, it can be seen that the obtained a12K4E-Ca mainly mediates the expression of mRNA in the spleen, and no obvious luciferase signal is seen in other organs including the thymus. Next, the effect of the PIL lipid structure on the expression efficiency and specificity of mRNA in the thymus was further explored. Compared with Am-Ka12K4, the expression efficiency of Am-Ka12K2 mRNA-LNP containing only 2 AIFA blocks is reduced in both the lung and thymus; on the basis of Am-Ka12K4, an additional amino acid including valine (V), serine (S), histidine (H) and tyrosine (Y) was introduced at the N-terminus, and Am-KVa12K4, Am-KSa12K4, Am-KHa12K4 and Am-KYa12K4 were obtained respectively. Among them, Am-KVa12K4 mRNA-LNP is mainly expressed in the lung, Am-KSa12K4 and Am-KYa12K4 are mainly expressed in the lung and thymus, while effective mRNA expression is observed in the liver, spleen, lung and thymus of Am-KHa12K4. Based on the above results, the inventors can conclude that the modification of the N-terminus of PIL with positively charged amino acids (including lysine and arginine) can significantly increase the expression of its mRNA-LNP in the thymus, and the delivery efficiency of PIL containing 4 AIFA blocks in the thymus is higher than that of PIL containing 2 AIFAs. In order to further explore whether the types of fatty chains and AIFA will affect the expression of mRNA-LNP in the thymus, the inventors synthesized e12K4 containing a hydroxyl-containing fatty chain, a12Dab4 containing Dab and Am-Ka12Dab4. In vivo results show that the addition of both the hydroxyl-containing fatty chain and Dab enhances the expression of mRNA-LNP in the thymus region. Quantitative data show that ( Figure 11 ), the average bioluminescence intensities of a12Dab4 containing the Dab block and Am-a12Dab4 in the thymus region reached 6×10^6 and 2×10^6 respectively. In addition, Am-KYa12K4 also mediates the effective expression of mRNA in the thymus, and its average bioluminescence intensity reached 1.2×10^6. The inventors further compared the specificity of mRNA expression of a12Dab4 and Am-KYa12K4 in the thymus ( Figure 12), it was found that although the expression intensity of a12Dab4 mRNA-LNP at the thymus was about 5 times that of Am-KYa12K4, the proportion of its mRNA expressed in the thymus was only 7.78%; while the thymic expression proportion of Am-KYa12K4 reached 66.4%, which was significantly superior to a12Dab4 in terms of the thymic expression specificity of mRNA.

[0132] Finally, the inventor selected the more specific Am-KYa12K4 lipid and combined it with Luc mRNA containing miRNA binding sites to further improve the thymic expression specificity of Am-KYa12K4 LNP. Considering that Am-KYa12K4 LNP is mainly expressed in the lungs in addition to being effectively expressed in the thymus, the inventor selected miR-126 with high expression in the lungs and designed Luc mRNA (miR126ts-Luc mRNA, Table 2) containing miR-126 binding sites (miR126ts, Table 1) for this miRNA ( Figure 13 ). The in vivo delivery results showed ( Figure 14 ), Am-KYa12K4 LNP encapsulating miR126ts-Luc mRNA could effectively mediate the expression of mRNA in the thymus, and its average bioluminescence intensity was about 3.2×10^5. Although this intensity was lower than that of Am-KYa12K4 mRNA-LNP without miR126ts, the introduction of the miR126ts site significantly reduced the expression of mRNA in the lungs. Therefore, the specificity of its thymic mRNA expression was further increased from 66.4% to 82.4%. The above results indicate that Am-KYa12K4 mRNA-LNP combined with miR126ts has a high mRNA expression selectivity for the thymus and has certain application potential for the development of mRNA therapies for treating thymus-related diseases.

[0133]

[0134]

[0135] Synthesizing novel organ-targeted ionizable lipids is one of the main strategies for developing extrahepatic organ-specific targeted mRNA-LNP. Currently, through rational design of lipid structures and high-throughput screening strategies in vivo, ionizable lipids that can specifically target organs such as the liver, spleen, lung, lymph node, and bone marrow have been reported. However, there is still no report on ionizable lipids that can highly specifically target the thymus.

[0136] In view of this current situation, in this project, the inventors synthesized a series of PILs based on artificially synthesized AIFA building blocks and natural amino acid building blocks using solid-phase peptide synthesis technology. Through in vivo mRNA delivery efficiency tests, it was found that: (1) PILs with positively charged amino acids (lysine and arginine) modified on the N-terminal side chain can mediate mRNA expression in the thymus with high selectivity; (2) PILs containing hydroxyl-containing aliphatic chains mediate mRNA expression in the thymus more efficiently than saturated alkane chains; (3) PILs containing Dab building blocks mediate mRNA expression in the thymus more efficiently than PILs containing K building blocks. Based on the above findings, the inventors selected the PIL lipid Am-KYa12K4 and its corresponding mRNA-LNP that can effectively and highly specifically deliver mRNA to the thymus for expression. By introducing miR-126 binding sites (miR126ts) into the mRNA sequence, the expression of Am-KYa12K4 mRNA-LNP in the lungs was reduced, further enhancing its expression specificity in the thymus. In this project, thymus-targeted mRNA-LNP has certain application potential for the development of mRNA therapies targeting thymus lesions; the design and structure-activity relationship of thymus-targeted ionizable lipid structures have certain guiding significance for the subsequent development of more efficient and safe thymus-targeted ionizable lipids in the field; the combined use strategy of targeted ionizable lipids and mRNA containing miRNA binding sites has certain reference value for further enhancing the organ-targeting specificity of mRNA-LNP.

Claims

1. A peptidyl ionizable lipid of the following formula I, in, o is an integer from 1 to 30, m and n are integers from 0 to 30, p is an integer from 0 to 10, X is independently O or S at each occurrence, A1 and A3 are independently hydrogen or alkyl groups at each occurrence. R2, R4, at each occurrence, independently represent a side group contained in an amino acid building block in an ionizable lipid, wherein R2 is a natural amino acid side group or an unnatural amino acid side group, wherein R4 is any amino acid side group having a pKa greater than or equal to about 6.0, A4 and A5 are hydrophobic tails and are independently of each other an optionally substituted saturated or unsaturated, linear or branched alkyl chain having 4 to 25 carbon atoms, wherein the alkyl chain optionally contains one or more linking groups L selected from an amide bond, an ester bond, a disulfide bond, a thioketal bond, an ether bond or a combination thereof, R6 represents the C-terminus of the peptidyl ionizable lipid, and R6 is a hydroxyl group or is modified with an amino group, an amino acid and / or other functional groups.

2. The peptidyl ionizable lipid according to claim 1, wherein R4 is a basic natural amino acid side group such as a lysine side group (-CH2CH2CH2CH2NH2), an arginine side group (-CH2CH2CH2NHC(NH)NH2) or a histidine side group (-CH2C3H3N2).

3. The peptidyl ionizable lipid according to claim 1, wherein the peptidyl ionizable lipid has the following formula II, 。 4. The peptidyl ionizable lipid according to any one of claims 1 to 3, wherein R2 is a natural amino acid side group, and the natural amino acid is preferably selected from serine, threonine, histidine, lysine, arginine, tyrosine, tryptophan and phenylalanine, more preferably R2 is a serine side group (-CH2OH), a histidine side group (-CH2C3H3N2) or a tyrosine side group (-CH2C6H4OH).

5. The peptidyl ionizable lipid according to any one of claims 1 to 4, wherein p is 1, 2, 3 or 4, preferably 2.

6. The peptidyl ionizable lipid according to any one of claims 1 to 5, wherein A4 or A5 is a hydrophobic tail substituted with a hydroxyl group, preferably A4 or A5 is -CH2CHOH(CH2) q CH2CH3, wherein q is an integer from 0 to 21.

7. The peptidyl ionizable lipid according to any one of claims 1 to 6, wherein o is an integer greater than 3, such as 3, 4, 5 or 6.

8. The peptidyl ionizable lipid according to any one of claims 1 to 7, wherein the peptidyl ionizable lipid is selected from: ， ， ， ， ， ， ， ， ， ， 。 9. A thymus-targeted lipid nanoparticle (LNP) comprising the peptidyl ionizable lipid according to any one of claims 1 to 8, optionally a helper lipid and optionally an active agent.

10. The LNP of claim 9, wherein the active agent is a polynucleotide containing one or more regulatory sequences that reduce the expression of the polynucleotide in a non-thymic organ, preferably the non-thymic 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, brainstem, cerebellum, spinal cord, eye, ear, tongue or skin, more preferably the non-thymic organ is the lung.

11. The LNP according to claim 10, wherein the one or more regulatory sequences include a miRNA binding site, preferably the miRNA binding site binds to a miRNA selected from the group consisting of miR-122, miR-133, miR-206, miR-208, miR-17-92, miR-126, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, miR-204, miR142, miR150 or a combination thereof, more preferably the miRNA binding site binds to let-7, miR-133, miR-126 or a combination thereof.

12. The LNP of claim 11, wherein the miRNA binding site comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99% identity to the sequence shown in SEQ ID NO:

1.

13. A pharmaceutical composition comprising the LNP according to any one of claims 9 to 12 and a pharmaceutically acceptable carrier.

14. Use of the LNP according to any one of claims 9 to 12 or the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating or preventing a disease, preferably a disease that is improved by targeted delivery of an active agent to the thymus.

15. Use of the peptide-based ionizable lipid according to any one of claims 1 to 8 in the preparation of thymus-targeted LNPs.

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