Fatty acid derivative for delivering nucleic acid substances and synthesis method thereof

Abstract

The invention provides a fatty acid derivative for delivering nucleic acid substances and a synthesis method thereof. The fatty acid derivative is used for modifying nucleic acid substances such as long-chain nucleic acid, oligonucleotide or double-chain small nucleic acid. The fatty acid derivative can be covalently or non-covalently bound with the nucleic acid substance, so that the in-vivo efficacy of the nucleic acid substance is enhanced, the side effect is reduced, and the storage stability is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology. The present invention provides a novel lipophilic compound and its derivatives (FADS) for modifying nucleic acid substances such as long-chain nucleic acids, oligonucleotides or double-stranded small nucleic acids. The lipophilic compound and its derivatives can be covalently or non-covalently bound to the nucleic acid substances, enhancing the efficacy of the nucleic acid substances in vivo, reducing side effects and improving storage stability. Background Art

[0002] Nucleic acids are carriers of genetic information in living organisms. It has been found that artificially designed nucleic acid nucleotide sequences can exhibit different functions in vivo: (1) They can express proteins or peptides in living organisms, and these proteins and peptides may have various physiological functions for preventing and treating diseases. (2) They can directly interact with the corresponding mRNA in living organisms, up-regulating or down-regulating the expression of downstream functional proteins or enzymes, or activating and inhibiting the activities of downstream functional proteins or enzymes. (3) They can act on pre-mRNA in living organisms, editing the generation of downstream mRNA, enabling the expression of mRNA of therapeutic proteins, enzymes or peptides, thereby treating diseases. Theoretically, they can regulate the expression of any gene, thus expanding their therapeutic application scope and potentially solving genetic diseases and other intractable diseases lacking effective drugs.

[0003] Compared with traditional drugs (such as small molecules, antibodies and proteins) that act at the protein level, drugs composed of nucleic acids have a simpler, more efficient and more bi-specific development process. Compared with DNA genome-level therapies, nucleic acid drugs can reduce the risks associated with gene integration in living organisms. Therefore, the therapeutic applications of nucleic acid drugs are more extensive and are expected to solve the treatment problems of genetic diseases and other intractable diseases.

[0004] However, nucleic acid substances are structurally unstable and are easily degraded by nucleases in blood and tissues. In addition, the hydrophilicity and negative charge of nucleic acids also hinder their uptake by cells through cell membranes. Exogenous nucleic acid molecules are immunogenic and will be cleared by the immune system in vivo.

[0005] Although chemical modifications have been made to individual nucleotides, such as base modification, sugar modification and phosphate modification, resulting in various nucleic acid substances, and nucleic acid sequence optimization techniques have also improved the stability of nucleic acids in vivo and reduced their immunogenicity to a certain extent, safely and effectively delivering nucleic acids (such as long-chain nucleic acids, oligonucleotides or double-stranded small nucleic acids) into living organisms remains the greatest challenge in nucleic acid applications.

[0006] Currently, the main delivery technologies of nucleic acid drugs on the market and in research include non-covalent carriers such as viral vectors, lipid nanoparticles (LNPs), and covalent carriers such as GalNAc (N-acetylgalactosamine).

[0007] Viral vectors have high transfection efficiency and are widely used in vaccines and cell therapies. Common viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. However, viral vectors also have limitations, including significant cytotoxicity, immunogenicity, and biosafety risks.

[0008] LNP delivery technology utilizes non-covalent assembly of lipid molecules to form nanoparticles. Its structure resembles that of a cell membrane. The positively charged amino groups in LNPs bind to negatively charged nucleic acids to form nanoparticles, enhancing the stability of mRNA molecules and enabling in vivo delivery of nucleic acids. When the mRNA / LNP reaches the cell membrane, cationic phospholipids trigger membrane fusion with the negatively charged cell membrane, leading to internalization into the cell. Lysosomal degradation creates a slightly acidic environment, causing some lipid protonation, disrupting the LNP bilayer structure and releasing the physiologically active mRNA. While LNP transfection efficiency is lower than that of viral vectors, it offers excellent biosafety and is a relatively mature technology. Similar technologies have been widely used in small molecule anti-tumor drugs, such as Doxil and Onivyde. Recently, LNPs have been successfully applied to nucleic acid-based drugs, such as the COVID-19 vaccine, including Moderna's mRNA-1273 and Pfizer-BioNTech's BNT162b2. However, LNPs for nucleic acid-based drugs suffer from poor storage stability. Therefore, LNP-delivered nucleic acid vaccines must be stored below -20°C and have a shelf life of only about one year.

[0009] GalNAc delivery technology is a covalent modification technology for nucleic acid drugs. It involves modifying a carbohydrate compound onto a nucleic acid molecule. The head of the structure contains three galactose groups, and the nucleic acid drug is covalently bound to the 3' end. GalNAc-RNA has a high affinity for asialoglycoprotein (ASGPR). ASGPR is a highly abundant, endocytic receptor found only in liver parenchymal cells that specifically recognizes and binds to GalNAc. After binding to ASGPR on the surface of liver parenchymal cells, GalNAc enters the cell through endocytosis, forming endosomes. This allows the nucleic acid drug to be carried inside the cell and released within the liver cell, achieving its therapeutic effect. GalNAc has been successfully applied in several small nucleic acid drugs currently marketed and under development. For example, Givlaari, inclisiran, and lumasiran, developed by Alnylam, are marketed for the treatment of various diseases. However, GalNAc requires a high dosage (250 mg / dose) and is prone to adverse reactions such as hepatotoxicity. Summary of the Invention

[0010] In order to solve the problems of the prior art, the present invention first provides a FADS derivative shown in Formula 1:

[0011]

[0012] Wherein:

[0013] R1 is a lipophilic fragment, and m is 1, 2 or 3;

[0014] R2 is an arbitrary (m + 1)-valent linking group;

[0015] R3 is a hydrophilic fragment;

[0016] H1 is selected from:

[0017] 1) A phosphate group, a thiophosphate group, a carboxyl group COOH, an amino group NH2, a hydroxyl group or a mercapto group, or a protected form or an activated form of the above groups;

[0018] 2) A phosphite group:

[0019]

[0020] 3) A cyclic linking group

[0021] where Cy is an optionally substituted five- or six-membered carbocyclic ring or a five- or six-membered heterocyclic ring containing one or two heteroatoms (oxygen atom or nitrogen atom), such as );

[0022] 4) An acyclic linking group

[0023]

[0024] wherein, G1 or G2 is independently selected from H, a hydroxyl protecting group such as DMTr, the phosphite group defined in 2) above, wherein CPG is a solid support, preferably controlled pore glass beads;

[0025] Any two of R1, R2, R3 and H1 are connected by an arbitrary group; preferably, they are connected by an amide group or an ether group.

[0026] In a specific embodiment of the present invention, R1 is a substituted or unsubstituted C 2-30 alkyl, C 2-30 alkenyl or C 2-30 alkynyl;

[0027] In a specific embodiment of the present invention, m is 1, and R2 is a substituted or unsubstituted C 1-10 alkylene, C 2-10 alkenylene or C 2-10 alkynylene;

[0028] In a specific embodiment of the present invention, m is 2, and R2 is C 1-10Alkylene, C 2-10 Alkenylene, C 2-10 Alkynylene or is derived from an amino acid residue having a reactive group in the side chain such as lysine, glutamic acid or aspartic acid, etc., for example:

[0029]

[0030] In a specific embodiment of the present invention, R3 is a group containing a polyethylene glycol fragment.

[0031] Preferably, R1 is derived from an optionally substituted fatty acid group, R1 is connected to R2 by an amide, and R1 is preferably:

[0032] Wherein n, n1, n2, n3, n4 and n5 are each independently an integer from 0 to 100; preferably, the sum of n or n1 - n5 is selected from integers of 4 - 30, and more preferably is an integer of 8 - 22.

[0033] Preferably, R2 is an optionally substituted C2 - C6 alkylene group, and the substituent group can be selected from OH, NH2 or C1 - C4 alkyl.

[0034] Preferably, R3 contains 2 - 20 ethoxy units, more preferably contains 4 - 10 ethoxy units; further preferably, every 2 or 3 ethoxy units in R3 are separated by an amide bond.

[0035] More preferably, R3 is:

[0036]

[0037] Wherein, n6 is an integer from 1 to 9, preferably an integer from 1 to 4 (specifically 1, 2, 3 or 4); preferably, R2 and R3 are connected by an amide bond.

[0038] Preferably, the FADS compound is a FADS monomer:

[0039]

[0040] Preferably, the FADS compound is a FADS-coupled solid support (FADS-CPG):

[0041]

[0042] The second aspect of the present invention provides a modified nucleotide or nucleic acid molecule (Formula II), including:

[0043]

[0044] Preferably is

[0045]

[0046] Preferably:

[0047]

[0048] Preferably:

[0049]

[0050]

[0051]

[0052] Wherein O in the phosphate group is optionally replaced by S.

[0053] Wherein X3 is a nucleotide or a nucleic acid molecule.

[0054] The nucleotide is selected from uridine nucleotide, thymidine nucleotide, cytidine nucleotide, 5-methylcytidine nucleotide, adenosine nucleotide or guanosine nucleotide.

[0055] The nucleic acid molecule is selected from small interfering RNA, microRNA, alternative spliceosome, single-stranded RNA, double-stranded RNA, antisense nucleic acid, aptamer, mRNA or DNA.

[0056] Preferably, the nucleic acid molecule has 2 or 3 consecutive modifications at the 5′ end or 3′ end, for example:

[0057]

[0058] In a preferred embodiment of the present invention, each nucleic acid molecule binds 1-6 (specifically: 1, 2, 3, 4, 5 or 6) of the above-mentioned compounds of formula I.

[0059] In a specific embodiment of the present invention, the nucleic acid molecule further comprises other modifications, including but not limited to the following modifications: locked nucleic acid modification, open-loop or non-locked nucleic acid modification, 2′-methoxyethyl modification, 2′-O-methyl modification, 2′-O-allyl modification, 2′-C-allyl modification, 2′-fluoro modification, 2′-deoxy modification, 2′-hydroxy modification or phosphorothioate backbone modification.

[0060] In a preferred embodiment of the present invention, the nucleic acid molecule is an siRNA molecule that inhibits the expression of a target gene, including a sense strand and an antisense strand that complementarily form a double-stranded region. The sense strand and / or the antisense strand comprise or consist of 15-25 nucleotides. The antisense strand is fully or partially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the target gene. The length of the double-stranded region is 15-25 bp, and at least one nucleotide in the siRNA molecule comprises a modification of a compound of Formula I.

[0061] The target genes include, but are not limited to: ANGPTL3, PCSK9, ApoB, ApoC, FVII, AGT, Lp(a), XDH, HSD17B13, SCD1, PNPLA3, HMGCR, etc.

[0062] The target genes also include genes encoding functional fragments of core proteins, viral polymerases, surface antigens, e-antigens, or X proteins in HAV, HBV, HCV, HDV, and HEV.

[0063] The third aspect of the present invention provides a pharmaceutical composition comprising the modified nucleotide or nucleic acid molecule of the second aspect.

[0064] The fourth aspect of the present invention provides the use of the above-mentioned modified nucleotide or nucleic acid molecule or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a physiological condition or disease caused by the expression of a specific gene.

[0065] The specific genes include, but are not limited to: ANGPTL3, PCSK9, ApoB, ApoC, FVII, AGT, Lp(a), XDH, HSD17B13, SCD1, PNPLA3, HMGCR, etc.

[0066] The specific genes also include genes encoding functional fragments of core proteins, viral polymerases, surface antigens, e-antigens, or X proteins in HAV, HBV, HCV, HDV, and HEV.

[0067] Furthermore, in the use provided by the present invention, the diseases are chronic liver diseases, hereditary transthyretin (hATTR) amyloidosis, hepatitis, liver fibrosis diseases, liver hyperplastic diseases, liver cancer, hypertension, and other dyslipidemia diseases.

[0068] The fifth aspect of the present invention provides a method for preventing and / or treating a physiological condition or disease caused by the expression of a specific gene, which comprises administering the above-mentioned modified nucleotide or nucleic acid molecule or the above-mentioned pharmaceutical composition to an individual in need.

[0069] The sixth aspect of the present invention provides a method for preparing 3'-modified FADS-siRNA, the method comprising the following steps: synthesizing siRNA using a solid-phase support, and then adding the above-mentioned FADS monomer; after the reaction is completed, cleaving the synthesized sequence from the support, and then annealing to obtain 3'-modified FADS-siRNA.

[0070] The present invention also provides a method for preparing 3'-modified FADS-ASO, the method comprising the following steps: synthesizing ASO using a solid-phase support, and then adding a FADS monomer to be linked to the 3'-end of the ASO; subsequently, cleaving the synthesized FADS-ASO from the support.

[0071] The present invention also provides a method for preparing FADS-siRNA-FADS modified at the 5'- and 3'-ends, the method comprising synthesizing siRNA using a modified solid-phase support (FADS-CPG), and then adding a FADS monomer; after the reaction is completed, cleaving the synthesized sequence from the support and annealing to obtain doubly modified FADS-siRNA-FADS.

[0072] The present invention also provides a method for preparing FADS-ASO modified at the 5'- and 3'-ends, the method comprising synthesizing ASO using a modified solid-phase support (FADS-CPG), and then adding a FADS monomer; cleaving the synthesized sequence from the support to obtain doubly modified FADS-ASO-FADS.

[0073] The seventh aspect of the present invention provides a composition comprising a nucleic acid molecule, the composition comprising the above-mentioned FADS compound, phospholipid and cholesterol.

[0074] In a specific embodiment of the present invention, the composition is a liposome or LNP.

[0075] The nucleic acid molecule includes small interfering RNA, microRNA, alternative spliceosome, single-stranded RNA, double-stranded RNA, antisense nucleic acid, nucleic acid aptamer, mRNA or DNA.

[0076] The term

[0077] is the covalent linkage site of the group.

[0078] The terms "optionally", "optionally", "optionally" or "optionally" mean that the subsequent described event or condition may but does not necessarily occur, and the description includes the case where the described event or condition occurs and the case where the described event or condition does not occur.

[0079] The term "oxo group" means that two hydrogen atoms at the same substitution position are replaced by the same oxygen atom to form a double bond.

[0080] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, including straight-chain or branched-chain groups containing 1-30 carbon atoms, preferably containing 4-28 carbon atoms (i.e., C 6-24 alkyl), more preferably containing 10-24 carbon atoms (C 10-18 alkyl).

[0081] Unless otherwise specified, the term "alkenyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond. The alkenyl can contain 2-30 carbon atoms, preferably containing 6-24 carbon atoms (i.e., C 6-24 alkenyl), more preferably containing 10-18 carbon atoms (C 10-18 alkenyl).

[0082] Unless otherwise specified, the term "alkynyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond. The alkynyl can contain 2-30 carbon atoms, preferably containing 6-24 carbon atoms (i.e., C 2-10 alkynyl).

[0083] “C 1-10 alkylene” refers to a divalent group formed by removing another hydrogen of C 1-10 alkyl, and can be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene and C 1-3 alkylene are preferred. The unsubstituted alkylene includes, but is not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), etc. Exemplary substituted alkylene, for example, alkylene substituted by one or more alkyl groups (methyl), includes, but is not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0084] “C 2-10 alkenylene” refers to removing C2-10 In some embodiments, C 2-4 Alkenylene is particularly preferred. Exemplary unsubstituted alkenylene groups include, but are not limited to, vinylene (-CH=CH-) and propenylene (e.g., -CH=CHCH2-, -CH2-CH=CH-). Exemplary substituted alkenylene groups, for example, alkenylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted ethylene (-C(CH3)=CH-, -CH=C(CH3)-), substituted propenylene (-C(CH3)=CHCH2-, -CH=C(CH3)CH2-, -CH=CHCH(CH3)-, -CH=CHC(CH3)2-, -CH(CH3)-CH=CH-, -C(CH3)2-CH=CH-, -CH2-C(CH3)=CH-, -CH2-CH=C(CH3)-), and the like.

[0085] “C 2-10 "Alkynylidene" refers to the removal of C 2-10 In some embodiments, C 2-4 Alkyne is particularly preferred. Exemplary alkynyl includes, but is not limited to, ethynyl (-C≡C-), substituted or unsubstituted propynyl (-C≡CCH2-), and the like.

[0086] As used in the present invention, "optionally substituted" means that the corresponding group is unsubstituted or substituted by a substituent group, and the substituent group includes hydroxyl, carboxyl, C1-4 straight chain or branched alkyl, amino, cyano, halogen, oxo, nitro, amide or oxo C1-4 straight chain or branched alkyl.

[0087] The term "fatty acid" refers to a carboxylic acid having an aliphatic chain, which may be saturated or unsaturated. That is, a fatty acid is composed of a hydrocarbon group composed of carbon and hydrogen linked to a carboxyl group, and the number of carbon atoms in the fatty acid may be 4-30, preferably 8-22 (including the carbon atoms of the carboxyl group), and more preferably 14-22. Examples of saturated fatty acids include caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), arachidic acid (C20) or behenic acid, etc.; examples of unsaturated fatty acids include monounsaturated fatty acids such as myristic acid (C14:1, cis-9), palmitic acid (C16:1, cis-9), oleic acid (C18:1, cis-9), trans-oleic acid (C18:1, trans-9), ricinoleic acid (C18:1, cis-9), etc., or polyunsaturated fatty acids such as linolenic acid, DHA and EPA, etc.

[0088] The term "group containing a polyethylene glycol fragment" refers to a subunit based on polyethylene glycol or a polyethylene glycol functional derivative, and the polyethylene glycol functional derivative includes α-amino-ω-carboxy-polyethylene glycol, etc.; in the present invention, "polyethylene glycol" includes a polymer obtained by polymerization of ethylene oxide, or a homogeneous compound obtained by a chemical reaction of a compound containing an ethoxy unit (such as NH2-CH2CH2O-CH2CH2O-CH2-COOH), that is, any number of ethoxy units in the "group containing a polyethylene glycol fragment" can be spaced or interrupted by an optional chemical group.

[0089] Beneficial technical effects

[0090] The present invention provides a novel nucleic acid delivery technology platform, the core of which is a new chemical entity called fatty acid derivative (FADS), and FADS can covalently or non-covalently bind to nucleic acid substances, thereby enhancing their stability and effectiveness. Description of the drawings

[0091] Figure 1 It is the synthesis process of the FADS-CPG solid-phase carrier.

[0092] Figure 2 It is the LC-MS spectrum of FADS.

[0093] Figure 3 It is the HPLC chromatogram of FADS.

[0094] Figure 4 It shows the HNMR results of FADS

[0095] Figure 5 It is a schematic diagram of FADS modification at the 5' end of nucleic acid.

[0096] Figure 6 It is the LC-MS spectrum of SS

[0097] Figure 7 It is the LC-MS spectrum of AS

[0098] Figure 8 It is the UPLC chromatogram of SS.

[0099] Figure 9 It is the UPLC chromatogram of AS.

[0100] Figure 10 It shows the uptake of FADS-siRNA by HepG2 cells.

[0101] Figure 11 It shows the interference efficiency of FADS-siRNA on intracellular PCSK9.

[0102] Figure 12 is the distribution ratio of the drug in different tissues.

[0103] Figure 13 is the synthesis process of the FADS monomer.

[0104] Figure 14 is a schematic diagram of the FADS modification at the 3'-end of the nucleic acid.

[0105] Figure 15 is a schematic diagram of the FADS modification at the 5'-end of the ASO.

[0106] Figure 16 shows a schematic diagram of the FADS modification at the 5'-end and 3'-end of the siRNA. Detailed implementation mode

[0107] The present invention provides a novel nucleic acid delivery technology platform, the core of which is a new chemical entity called fatty acid derivative (FADS). FADS can covalently or non-covalently bind to nucleic acid substances, thereby enhancing their stability and effectiveness. The nucleic acids mentioned here include long-chain nucleic acids, oligonucleotides or double-stranded small nucleic acids. The present invention details the structural characteristics, structure confirmation, synthesis method of FADS and the basic method of binding to nucleic acids. In addition, the present invention also conducts comprehensive comparative experiments on the LNP and GalNac systems, including in vitro cell targeting experiments, in vitro nucleic acid potency experiments, in vivo tissue distribution experiments and in vivo target mRNA knockout experiments. The experimental results show that FADS can improve the stability and effectiveness of nucleic acids in vitro and in vivo. At the same dose, the target tissue distribution of FADS is significantly higher than that of LNP and GalNac carriers, and the knockout effect on target mRNA is significantly better than that of LNP and GalNac technologies.

[0108] To achieve the object of the present invention, the technical solution of the present invention first provides a fatty acid derivative (FADS) for delivering nucleic acid substances and its synthesis method.

[0109] The structure of the fatty acid derivative (FADS) for delivering nucleic acid substances is X1:

[0110]

[0111] Wherein:

[0112] R1 is selected from the following group:

[0113] R2 is selected from the following group:

[0114] R3 is

[0115] Wherein, n, n1, n2, n3, n4 and n5 are each independently an integer from 0 to 100; preferably, n or the sum of n1 - n5 is selected from integers from 4 to 30, and more preferably from 8 to 22.

[0116] The present invention also provides a FADS-linked nucleic acid, wherein the FADS is as described above, and the structure of the FADS-linked nucleic acid is X1-X2-X3, as follows:

[0117]

[0118] Wherein X1 is FADS, X2 is a linking moiety, and X3 is a nucleic acid.

[0119] FADS can be linked to the 5′ and 3′ ends of nucleic acid X3, or can be linked to both ends simultaneously.

[0120]

[0121] FADS can modify nucleic acids, such as oligonucleotides, siRNA, shRNA, mRNA, and DNA.

[0122] The composition of nucleic acid X3 delivered by FADS includes ribonucleic acid (RNA), deoxyribonucleic acid (DNA), nucleotide analogs, and nucleotides with various modifications.

[0123] In the technical solution of the present invention, the structure of R3 in FADS is represented as follows:

[0124]

[0125] Wherein, n6 is an integer from 1 to 9, preferably an integer from 1 to 4.

[0126] In the technical solution of the present invention, R1 of FADS can be a saturated fatty acid having the following structure:

[0127]

[0128] Wherein, n is any integer between 1 and 100.

[0129] Alternatively, R1 can be a carboxylic acid derivative of a saturated fatty acid, and its representative structure is as follows:

[0130]

[0131] Wherein, n can be any integer between 1 and 100.

[0132] Another option for R1 is an amino derivative of a saturated fatty acid, and its representative structure is as follows:

[0133]

[0134] It can be any integer between 1 and 100.

[0135] R1 can be an unsaturated fatty acid, and its structure is represented as:

[0136]

[0137] Among them, n1 is any integer from 1 to 100; n2 is any integer from 1 to 100; n3 is any integer from 0 to 100; n4 is any integer from 0 to 100; n5 is any integer from 0 to 100; preferably, n or the sum of n1 - n5 is selected from integers of 4 - 30, and more preferably integers of 8 - 22.

[0138] R1 can be a carboxylic acid derivative of an unsaturated fatty acid, represented as:

[0139]

[0140] Among them, n1 is any integer from 1 to 100; n2 is any integer from 1 to 100; n3 is any integer from 0 to 100; n4 is any integer from 0 to 100; n5 is any integer from 0 to 100; preferably, n or the sum of n1 - n5 is selected from integers of 4 - 30, and more preferably integers of 8 - 22..

[0141] R1 can be an amino derivative of an unsaturated fatty acid, represented as:

[0142]

[0143] Among them, n1 is any number from 1 to 100; n2 is any number from 1 to 100; n3 is any number from 0 to 100; n4 is any number from 0 to 100; n5 is any number from 0 to 100; preferably, n or the sum of n1 - n5 is selected from integers of 4 - 30, and more preferably integers of 8 - 22.

[0144] In the technical solution of the present invention, R2 can be represented as:

[0145]

[0146] In the technical solution of the present invention, the FADS for delivering nucleic acid substances as described in the present invention can modify nucleic acids through solid-phase synthesis. Specifically, X1 fatty acid derivatives are applied to nucleic acid synthesis on a solid-phase carrier. The structure of the solid-phase carrier is as follows:

[0147]

[0148] Among them, R1, R2 and R3 are as described above.

[0149] In this example, the synthesis steps of a solid-phase carrier of a fatty acid derivative (FADS) for delivering nucleic acid substances are provided.

[0150] The FADS for transporting nucleic acid substances according to the present invention can be modified on nucleic acids through solid-phase synthesis. Specifically, modification is carried out using an X1 fatty acid derivative as a synthesis monomer. The structure of the FADS monomer is as follows:

[0151]

[0152] Among them, R1, R2 and R3 are as described above.

[0153] The present invention also provides a method for synthesizing FADS, which includes the following steps:

[0154] Step (1): Intermediate 1 is obtained by reacting a fatty acid with methyl 4-aminobutyrate,

[0155] Step (2): Intermediate 2 is obtained by hydrolyzing Intermediate 1,

[0156] Step (3): Intermediate 3 is obtained by reacting Intermediate 2 with (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}tetrahydro-1H-pyrrol-3-ol), and

[0157] Step (4): FADS is obtained by reacting Intermediate 3 with succinic anhydride.

[0158] In this example, the synthesis steps of a monomer of a fatty acid derivative (FADS) for delivering nucleic acid substances are provided.

[0159] FADS can be used for covalent and non-covalent modification of nucleic acids. That is to say, FADS is for nucleic acids with covalent or non-covalent modification.

[0160] The nucleic acids delivered by FADS can be used for the treatment of in vivo diseases, and the administration methods include injection, oral administration and topical administration.

[0161] The present invention also provides the application of FADS, FADS-linked nucleic acids, FADS monomers or solid-phase carriers in the treatment of in vivo diseases, wherein the disease is a genetic disease.

[0162] In one embodiment, the present invention provides a synthetic method for preparing a solid-phase carrier modified with fatty acid derivatives (FADS). The method comprises the following steps: Intermediate 1 is obtained from the reaction of stearic acid, Intermediate 2 is obtained from the hydrolysis and further reaction of Intermediate 1, Intermediate 3 is generated by the continuous reaction of Intermediate 2, Intermediate 3 reacts with succinic anhydride to obtain Intermediate 4, and Intermediate 4 is coupled with amino-functionalized controlled pore glass beads to obtain a solid-phase carrier with a fatty acid derivative side chain for subsequent nucleic acid synthesis.

[0163]

[0164] Intermediate 1: 4-stearoylaminobutyric acid

[0165] Intermediate 2: 10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracosanoic acid

[0166] Intermediate 3: N-((1-(2S,4R)-2-((bis(4-methoxyphenyl)phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-1,10,19-trioxo-3,6,12,15-tetraoxa-9,18-diazabicyclo[22.2.2]docos-22-yl)stearamide

[0167] Intermediate 4: 4-((3R,5S)-5-((bis(4-methoxyphenyl)phenyl)methoxy)methyl)-1-(10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontanoyl)pyrrolidin-3-yl)oxy)-4-oxobutyric acid

[0168] In another embodiment, the present invention further provides a synthetic method for preparing 5'-modified FADS-siRNA, which comprises the following steps: using the solid-phase carrier FADS-CPG to synthesize siRNA, and then cleaving the synthesized sequence from the carrier and annealing it to obtain FADS-siRNA.

[0169] In a further embodiment, the present invention also provides a synthetic method for preparing 5'-modified FADS-ASO, which comprises the following steps: using the modified solid-phase carrier FADS-CPG to synthesize ASO, and then cleaving the synthesized FADS-ASO from the carrier.

[0170] In another embodiment, the present invention further provides a synthetic method for preparing fatty acid derivative (FADS) monomers, which comprises the following steps: stearic acid reacts to obtain Intermediate 1, Intermediate 1 undergoes hydrolysis and further reaction to obtain Intermediate 2. Intermediate 2 continues to react to generate Intermediate 3 and reacts with N,N-diisopropylethylamine to generate monomer 4. The specific synthesis steps are shown in the following formula.

[0171]

[0172] Intermediate 1: 4-Stearamidobutyric acid

[0173] Intermediate 2: 10,19,24-Trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracosanoic acid

[0174] Intermediate 3: N-((1-(2S,4R)-2-((Bis(4-methoxyphenyl)methyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)-1,10,19-trioxo-3,6,12,15-tetraoxa-9,18-diazabicyclo[22.2.2]docosan-22-yl)stearamide

[0175] Monomer 4: (3R,5S)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracosanoyl)pyrrolidin-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0176] In another embodiment, the present invention further provides a method for preparing 3'-modified FADS-siRNA. The method comprises the following steps: synthesizing siRNA using a solid support, and then adding FADS monomers; after the reaction is completed, cleaving the synthesized sequence from the support, and then annealing to obtain 3'-modified FADS-siRNA.

[0177] In another embodiment, the present invention provides a method for preparing 3'-modified FADS-ASO. The method comprises the following steps: synthesizing ASO using a solid support, and then adding FADS monomers to be linked to the 3'-end of the ASO. Subsequently, cleaving the synthesized FADS-ASO from the support.

[0178] In another embodiment, the present invention further provides a method for synthesizing FADS-siRNA-FADS modified at the 5'- and 3'-ends. The method comprises synthesizing siRNA using a solid support (FADS-CPG), and then adding FADS monomers. After the reaction is completed, cleaving the synthesized sequence from the support, and annealing to obtain doubly modified FADS-siRNA-FADS.

[0179] In another embodiment, the present invention provides a method for synthesizing FADS-ASO modified at the 5'- and 3'-ends. The method comprises synthesizing ASO using a solid support (FADS-CPG), and then adding FADS monomers. Cleaving the synthesized sequence from the support to obtain doubly modified FADS-ASO-FADS.

[0180] In addition, the present invention provides a method for preparing a nucleic acid non-covalent delivery platform using FADS. The method includes mixing FADS, phospholipids, and cholesterol to prepare liposomes containing nucleic acids.

[0181] Examples

[0182] Example 1

[0183] The synthesis conditions are applicable to all the listed compounds. Taking stearic acid as an example, the synthesis method of FADS-CPG is as Figure 1 shown:

[0184] The specific synthesis steps are as follows: Using stearic acid as the starting material, reacting with 4-aminobutyl methacrylate, hydrolyzing to obtain intermediate 1, and further reacting to obtain intermediate 2. Intermediate 2 reacts with (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidin-3-ol to form intermediate 3. Intermediate 3 reacts with succinic anhydride to form intermediate 4 (FADS). Intermediate 4 is coupled with amino-modified controlled pore glass beads to obtain a solid-phase carrier with a fatty acid derivative side chain, preparing for subsequent nucleic acid synthesis.

[0185] Intermediate 1: 4-stearamidobutyric acid

[0186] Intermediate 2: 10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracosanoic acid

[0187] Intermediate 3: N-((1-(2S,4R)-2-(bis(4-methoxyphenyl)methyl)oxymethyl)-4-hydroxypyrrolidin-1-yl)-1,10,19-trioxo-3,6,12,15-tetraoxa-9,18-diazabicyclo[22.2.2]docosane-22-yl)stearamide

[0188] Intermediate 4: 4-((3R,5S)-5-(bis(4-methoxyphenylphenyl)methoxy)methyl)-1-(10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetracontanyl)pyrrolidin-3-yl)oxy)-4-oxobutyric acid

[0189] The molecular weight of the synthesized product 4 was confirmed by LC-MS. The target molecular weight was 1161.49, and the strongest signal was observed at m / z = 1159.3 [M-2H] ( Figure 2 ), confirming the correct molecular weight of the product.

[0190] The purity of the product FADS was preliminarily evaluated by high performance liquid chromatography (HPLC). The column used was XBridge Shield RP 18 (150 mm*4.6 mm, 5 μm), the mobile phase was solvent A: 0.01 M NH4HCO3 aqueous solution, solvent B: acetonitrile, the flow rate was set at 1.5 mL / min, and the column temperature was maintained at 40°C. The HPLC results showed a well-separated peak at 8.898 minutes ( Figure 3 ), indicating that the product has high purity.

[0191] The structure of FADS was determined by using CDCl3 as solvent at 400 MHz. 1 H-NMR (nuclear magnetic resonance) spectroscopy confirmed that the NMR data are as follows ( Figure 4 ): δ=8.10-8.09 (multiple peak, 1H), 7.90-7.87 (triplet, J=12Hz, 1H), 7.74-7.71 (multiple peak, 2H), 7.34-7.27 (multiple peak, 8H), 6.84-6.79 ( Multiplet, 4H), 6.49-6.41 (multiplet, 1H), 5.32-5.23 (multiplet, 1H), 4.44-4.22 (multiplet, 2H), 4.00-3.97 (multiplet, 3H), 3.80-3.78 (multiplet, 6H), 3.67-3.55 (multiplet, 13H), 3.51-3.43 (multiplet, 5H), 3.27-3.20 (multiplet, 3H), 2.67-2.47 (multiplet, 6H), 2.30-2.23 (multiplet, 4H), 2.18-2.12 (multiplet, 3H), 1.82-1.75 (multiplet, 2H), 1.60-1.56 (multiplet, 2H), 1.25 (singlet, 28H), 0.89-0.86 (triplet, J=6.6 Hz, 3H). These NMR data confirmed that the product obtained by this synthesis method has the correct structure.

[0192] Controlled pore glass (CPG) beads were washed five times with an appropriate amount of dichloromethane, and the solvent was evaporated under reduced pressure. Intermediate 4, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 4-dimethylaminopyridine (DMAP), and N-ethyldiisopropylamine (DIPEA) were added to a plastic reaction vessel and stirred for 3-5 minutes. CPG beads were then added, the mixture sealed, and stirred at 45°C for 6 days. After filtration, the beads were washed sequentially with anhydrous N,N-dimethylformamide and anhydrous dichloromethane until thin-layer chromatography (TLC) showed no residual starting material. The solid was then dried under vacuum to obtain fatty acid derivative-functionalized CPG (FADS-CPG), which served as a solid support for subsequent nucleic acid synthesis.

[0193] Example 2: Synthesis of 5'-labeled FADS-siRNA

[0194] Using a twelve-channel nucleic acid synthesizer (HJ-12-20-0), the siRNA sequence targeting human PCSK9 with Cy5 fluorescence labeling was synthesized on the FADS-CPG solid support (SS: 5'-Cy5CUAGACCUGUUUUGCUUUUGU-FADS-3', AS: 5'-ACAAAAGCAAAACAGGUCUAGAA-3'). The synthesis steps are as Figure 5 shown.

[0195] The molecular weights of the two single strands were determined by LC-MS. The theoretical molecular weight of the sense strand (SS) was 8031.7, and the theoretical molecular weight of the antisense strand (AS) was 7431.6. The actual LC-MS results were 8031.5 SS( Figure 6 ) and 7432.7 AS( Figure 7 ), confirming the accuracy of the molecular weights. The purity of SS and AS was evaluated using UPLC( Figure 8 , 9 ). The results showed good separation, with strong signal peaks appearing for both strands. The purity of SS was 98.58%, and the purity of AS was 97.35%.

[0196] Example 3: Flow cytometry evaluation of FADS-siRNA uptake by HepG2 hepatocytes

[0197] HepG2 hepatocytes were seeded in 12-well plates and then transfected with Cy5-labeled FADS-siRNA at a concentration of 500 ng / ml. Untreated cells, cells transfected with siRNA alone, cells transfected with siRNA + Lipofectamine 3000, and cells transfected with GalNac-siRNA were used as controls. After 6 hours of transfection, the medium was removed, and the cells were collected for flow cytometry analysis to measure the cell fluorescence intensity.

[0198] As Figure 10 shown, the positive transfection rate of the GalNac-siRNA group was 24.2 ± 4.17%, while the positive transfection rate of the FADS-siRNA group was 40.93 ± 5.98%, which was 1.69 times that of the GalNac-siRNA group (P < 0.01). No significant differences were observed among the siRNA, siRNA + Lipofectamine 3000, and GalNac-siRNA groups, indicating that FADS-siRNA exhibited better transfection efficiency in HepG2 hepatocytes.

[0199] Example 4: RT-qPCR evaluation of the interference of FADS-siRNA on PCSK9

[0200] HepG2 hepatocytes were seeded in 12-well plates and then transfected with Cy5-labeled FADS-siRNA at a concentration of 500 ng / ml. Untreated cells, cells transfected with siRNA alone, cells transfected with siRNA + Lipofectamine 3000, and cells transfected with GalNac-siRNA were used as controls. After 24 hours of transfection, the medium was removed, and cellular RNA was extracted for RT-qPCR analysis to determine the PCSK9 mRNA level.

[0201] As Figure 11 shown, the knockdown efficiency of siRNA for PCSK9 was 2.96%, that of siRNA + Lipofectamine 3000 reached 15.4%, that of GalNac-siRNA reached 46.9%, and that of FADS-siRNA reached 56.42%. The knockdown efficiency of FADS-siRNA was higher than that of other groups, indicating that FADS-siRNA exhibited a better interference effect on PCSK9 mRNA.

[0202] Example 5: Fluorescence imaging to evaluate the distribution of FADS-siRNA in mouse tissues

[0203] Cy5-labeled PCSK9 siRNA was synthesized. Nine male C57BL / 6 mice weighing 20 g were divided into 3 groups: siRNA, GalNac-siRNA, and FADS-siRNA, with 3 mice in each group. Each mouse was subcutaneously injected with its respective siRNA at a dose of 0.01 nmol. After 48 hours of injection, the mice were euthanized, and the heart, liver, spleen, lung, and kidney tissues were dissected for fluorescence imaging, and then the fluorescence intensity was statistically calculated.

[0204] In the FADS-siRNA group, a higher amount of the drug ( Figure 12 ) was found in the liver compared with the other two groups, indicating that FADS-siRNA exhibited better liver targeting efficiency.

[0205] Example 6: In vivo knockdown evaluation of FADS-siRNA

[0206] Twenty-five C57BL / 6 mice (equal numbers of males and females) weighing 20 g were selected for the study and divided into 5 groups: normal saline control group, siRNA group, GalNac-siRNA group, high-dose FADS-siRNA group, and low-dose FADS-siRNA group, with 5 mice in each group. The mice in the FADS-siRNA groups were subcutaneously injected with the corresponding drugs. The dose of the high-dose FADS-siRNA group was 5 mg / kg, and that of the low-dose FADS-siRNA group was 2 mg / kg. After 1 week, mouse blood samples were collected, and the level of hPCSK9 in mouse serum was detected by ELISA.

[0207] Example 7: Synthesis of FADS monomer

[0208] The synthesis conditions are applicable to all listed compounds. Taking stearic acid as an example, the synthesis steps of FADS monomer are as follows: Figure 13 shown.

[0209] The specific synthesis steps are as follows: using stearic acid as a starting material, 4-aminobutyric acid methyl ester reacts to obtain intermediate 1; intermediate 1 undergoes hydrolysis and other reactions to obtain intermediate 2, intermediate 2 further reacts to generate intermediate 3, and intermediate 3 reacts with N,N-diisopropylethylamine to obtain monomer 4 that can be used for nucleic acid synthesis.

[0210] Intermediate 1: 4-Stearamidobutyric acid

[0211] Intermediate 2: 10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triaza-tetracosanoic acid

[0212] Intermediate 3: N-(1-(2S,4R)-2-(bis(4-methoxyphenylphenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-1,10,19-trioxa-3,6,12,15-tetraoxa-9,18-diazabehen-22-yl)stearamide

[0213] Monomer 4: (3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazaheptatetradecanoyl)pyrrolidin-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0214] Example 8: Synthesis of 3'-modified FADS-siRNA

[0215] Using CPG as a solid phase support, a siRNA sequence targeting human PCSK9 (SS: 5'-Cy5CUAGACCUGUUUUGCUUUUGU-FADS-3', AS: 5'-ACAAAAGCAAAACAGGUCUAGAA-3') was synthesized on a 12-channel nucleic acid synthesizer (HJ-12-20-0). After RNA synthesis, FADS monomers were introduced for modification, and the modified FADS-siRNA was then cleaved from the solid phase support. The synthesis steps are as follows: Figure 14 shown.

[0216] Example 9: Synthesis of 5'-modified FADS-ASO

[0217] Using a 12-channel nucleic acid synthesizer (HJ-12-20-0), the ASO sequence (5'-CAAAAAAGTGTTCCCTTTTCAAGTT-3') targeting human AGT was synthesized with FADS-CPG as the carrier. The modified FADS-ASO was cleaved from the solid-phase carrier, and the synthesis steps are as Figure 15 shown.

[0218] Example 10: Synthesis of FADS-siRNA-FADS with dual-terminal modification.

[0219] Using FADS-CPG as the solid-phase carrier, the siRNA sequence (SS: 5'-Cy5CUAGACCUGUUUUGCUUUUGU-FADS-3', AS: 5'-AAAAGCAAAACAGGUCUAGAA-3') targeting human PCSK9 with Cy5 fluorescence labeling was synthesized on a 12-channel nucleic acid synthesizer (HJ-12-20-0). After the RNA synthesis was completed, FADS monomers were added for modification, and then the siRNA was cleaved from the solid-phase carrier. After annealing, FADS-siRNA-FADS with dual-terminal modification was obtained, and the synthesis steps are as Figure 16 shown.

[0220] Although the present invention has been described in detail through general descriptions and specific examples, it is obvious to those skilled in the art that several modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements that do not depart from the spirit of the present invention are within the scope of the present invention.

[0221] Industrial Applicability

[0222] The present invention provides a new nucleic acid delivery technology platform, the core of which is a new chemical entity called fatty acid derivative (FADS). FADS can covalently or non-covalently bind nucleic acid substances, thereby enhancing their stability and effectiveness, and has good economic value and application prospects.

Claims

1. The FADS derivative shown in Formula 1: Where: R1 is a lipophilic fragment, and m is 1, 2 or 3; R2 is an arbitrary (m + 1)-valent linking group; R3 is a hydrophilic fragment; H1 is selected from: 1) A phosphate group, a thiophosphate group, a carboxyl group COOH, an amino group NH2, a hydroxyl group or a mercapto group, or a protected form or activated form of the above groups; 2) A phosphite group: 3) A cyclic linking group where Cy is an optionally substituted five- or six-membered carbocyclic ring or a five- or six-membered heterocyclic ring containing one or two heteroatoms (such as an oxygen atom or a nitrogen atom), for example 4) An acyclic linking group Among them, G1 or G2 is independently selected from H, a hydroxyl protecting group such as DMTr, the phosphityl group defined in 2) above, wherein CPG is a solid support, preferably controlled pore glass beads; Any two of R1, R2, R3 and H1 are linked by an arbitrary group; preferably, they are linked by an amide group or an ether group.

2. The FADS derivative according to claim 1, wherein R1 is a substituted or unsubstituted C 2-30 alkyl group, C 2-30 alkenyl group or C 2-30 alkynyl group; Preferably, R1 is derived from an optionally substituted fatty acid group, R1 is linked to R2 by an amide, and R1 is preferably: Where n, n1, n2, n3, n4 and n5 are each independently an integer from 0 to 100; preferably, n or the sum of n1 - n5 is selected from integers from 4 to 30, and more preferably from 8 to 22.

3. The FADS derivative according to claim 1, wherein m is 1 and R2 is a substituted or unsubstituted C 1-10 alkylene, C2-10 alkenylene or C 2-10 alkynylene; preferably, R2 is an optionally substituted C 2-6 alkylene, and the substituent group may be selected from OH, NH2 or C 1-4 alkyl.

4. The FADS derivative according to claim 1, wherein m is 2 and R2 is C 1-10 subalkyl, C 2-10 subalkenyl, C 2-10 subalkynyl or is a residue derived from an amino acid having a reactive group in the side chain, such as lysine, glutamic acid or aspartic acid, etc. For example:

5. The FADS derivative according to claim 1, wherein R3 is a group containing a polyethylene glycol fragment; Preferably, R3 contains 2 - 20 ethoxy units, more preferably, it contains 4 - 10 ethoxy units; further preferably, every 2 or 3 ethoxy units in R3 are separated by an amide bond. More preferably, R3 is: Among them, n6 is an integer from 1 to 9, preferably an integer from 1 to 4; Preferably, R2 and R3 are linked by an amide bond.

6. The FADS derivative according to claim 1, The FADS compound is a FADS monomer: Or The FADS compound is a solid-phase carrier conjugated with FADS (FADS-CPG):

7. A modified nucleotide or nucleic acid molecule, including: Preferably Preferably: Preferably: Wherein O in the phosphate group is optionally replaced by S; Where X3 is a nucleotide or nucleic acid molecule; Where R1, R2, R3 or m is as described in any one of claims 1 - 6.

8. The modified nucleotide or nucleic acid molecule according to claim 7, The nucleotide is selected from uridine nucleotide, thymidine nucleotide, cytidine nucleotide, 5-methylcytidine nucleotide, adenosine nucleotide or guanosine nucleotide; The nucleic acid molecule is selected from small interfering RNA, microRNA, alternative spliceosome, single-stranded RNA, double-stranded RNA, antisense nucleic acid, aptamer, mRNA or DNA; Preferably, the nucleic acid molecule further contains other modifications, including but not limited to the following modifications: locked nucleic acid modification, open-loop or non-locked nucleic acid modification, 2'-methoxyethyl modification, 2'-O-methyl modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-fluoro modification, 2'-deoxy modification, 2'-hydroxy modification or thiophosphate backbone modification; Preferably, the nucleic acid molecule is an siRNA molecule that inhibits the expression of a target gene, including a sense strand and an antisense strand that complementarily form a double-stranded region. The sense strand and / or the antisense strand comprises 15-25 nucleotides or consists of 15-25 nucleotides. The antisense strand is fully or partially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of the target gene. The length of the double-stranded region is 15-25 bp, and at least one nucleotide in the siRNA molecule contains a modification of a compound of Formula I; the target gene includes, but is not limited to: ANGPTL3, PCSK9, ApoB, ApoC, FVII, AGT, Lp(a), XDH, HSD17B13, SCD1, PNPLA3, HMGCR, etc.; the target gene also includes genes encoding functional fragments of core proteins, viral polymerases, surface antigens, e-antigens, or X proteins in HAV, HBV, HCV, HDV, and HEV.

9. The modified nucleotide or nucleic acid molecule according to claim 7, The nucleic acid molecule has 2 or 3 consecutive modifications at the 5′ end or the 3′ end, for example: Preferably, each nucleic acid molecule binds 1-6 (specifically: 1, 2, 3, 4, 5, or 6) of the above-mentioned compounds of Formula I.

10. A pharmaceutical composition comprising the modified nucleotide or nucleic acid molecule according to any one of claims 7-9.

11. Use of the modified nucleotide or nucleic acid molecule according to any one of claims 7-9 or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a physiological condition or disease caused by the expression of a specific gene; Preferably, the specific gene includes, but is not limited to: ANGPTL3, PCSK9, ApoB, ApoC, FVII, AGT, Lp(a), XDH, HSD17B13, SCD1, PNPLA3, HMGCR, etc. Preferably, the specific gene also includes genes encoding functional fragments of core proteins, viral polymerases, surface antigens, e-antigens, or X proteins in HAV, HBV, HCV, HDV, and HEV. Preferably, the disease is chronic liver disease, hereditary transthyretin (hATTR) amyloidosis, hepatitis, liver fibrosis disease, liver hyperplastic disease, liver cancer, hypertension, and other dyslipidemia diseases.

12. A method for preventing and / or treating a physiological condition or disease caused by the expression of a specific gene, which comprises administering to an individual in need the modified nucleotide or nucleic acid molecule according to any one of claims 7-9 or the pharmaceutical composition according to claim 10.

13. A composition comprising a nucleic acid molecule, the composition comprising the FADS derivative, phospholipid, and cholesterol according to any one of claims 1-6; Preferably, the composition is a liposome or LNP; Preferably, the nucleic acid molecule includes small interfering RNA, microRNA, alternative spliceosome, single-stranded RNA, double-stranded RNA, antisense nucleic acid, nucleic acid aptamer, mRNA, or DNA.

14. A method for preparing a nucleic acid molecule with 3′FADS modification and / or 5′ modification of FADS, comprising the following steps: Synthesize nucleic acid molecules using an optionally FADS-modified solid support, and then optionally add FADS monomers; after the reaction is completed, cleave the synthesized sequence from the support and optionally anneal to obtain a modified nucleic acid molecule; provided that at least one of the FADS-modified solid support or the added FADS monomer is selected; the FADS-modified solid support and the FADS monomer have the structures described in claim 6; Preferably, the method is a method for preparing 3'-modified FADS-siRNA, and the method comprises the following steps: synthesize siRNA using a solid support, and then add the above-mentioned FADS monomers; after the reaction is completed, cleave the synthesized sequence from the support, and then anneal to obtain 3'-modified FADS-siRNA; Preferably, the method is a method for preparing 3'-modified FADS-ASO, and the method comprises the following steps: synthesize ASO using a solid support, and then add FADS monomers to be linked to the 3' end of the ASO; subsequently, cleave the synthesized FADS-ASO from the support; Preferably, the method is a method for preparing FADS-siRNA-FADS modified at both the 5' and 3' ends, and the method comprises synthesizing siRNA using a modified solid support (FADS-CPG), and then adding FADS monomers; after the reaction is completed, cut the synthesized sequence from the support and anneal to obtain doubly modified FADS-siRNA-FADS; Preferably, the method is a method for preparing FADS-ASO modified at both the 5' and 3' ends, and the method comprises synthesizing ASO using a solid support (FADS-CPG), and then adding FADS monomers; cut the synthesized sequence from the support to obtain doubly modified FADS-ASO-FADS.

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