AU2023202944A1

AU2023202944A1 - A nucleic acid targeting proprotein convertase subtilisin/kexin 9 and uses thereof

Info

Publication number: AU2023202944A1
Application number: AU2023202944A
Authority: AU
Inventors: Weiwen Jiang; Jimmy Xibai Tang; Dong Yu
Original assignee: Synerk Biotech Ltd
Current assignee: Synerk Biotech Ltd
Priority date: 2023-05-11
Filing date: 2023-05-11
Publication date: 2024-11-28

Abstract

The present disclosure relates to the technical field of biotechnology, and discloses a nucleic acid targeting proprotein convertase subtilisin/kexin 9 and uses thereof. The nucleic acid provided by the present disclosure can effectively inhibit the expression of PCSK9, thereby being capable of treating and/or preventing hyperlipidemia related diseases.

Description

A NUCLEIC ACID TARGETING PROPROTEIN CONVERTASE

SUBTILISIN/KEXIN 9 AND USES THEREOF

FIELD

[0001] The present disclosure relates to the technical field of biotechnology, in particular to a nucleic acid targeting proprotein convertase subtilisin/kexin 9 and uses thereof.

BACKGROUND

[0002] The proprotein convertase subtilisin/kexin 9 (PCSK9, also known as FH3, FHCL3, HCHOLA3, LDLCQ 1, NARC-1, NARC1, PC9) is the 9th member of the proprotein convertase

subtilysin family. The human PCSK9 gene, located at chromosome 1p3213, is approximately

22 kb in length and contains 12 exons, encodes a pro-keratin convertase including 692 amino

acid residues, and is formed by connecting a signal peptide (SP1-30), a pre-structural domain

(31-152), a catalytic domain (153-452) and a carboxy-terminal domain (526-692) sequentially;

it is mainly expressed in liver, kidney and small intestine (Stoekenbroek R et al., Nat Rev

Endocrinol. 15:52-62, 2019).

[0003] After secretion of the PCSK9 into the extracellular, the PCSK9 binds with the low

density lipoprotein (LDL) receptor on the cell surface, the PCSK9-LDL receptor complex enters

into lysosome for degradation, resulting in the decreased LDL receptor on the cell surface, i.e.,

the PCSK9 level is inversely correlated with the LDL receptor (Park S et al. J Biol chem. 279:

50630-50638, 2004; Shapiro M et al. Circulation Res. 122:1420-1438). Studies have shown

that loss-of-mutation function of PCSK9 gene may lead to a significant decrease in LDL-C

level and the incidence of coronary heart disease in different human species (Berge K et al.

Arterioscler Thromb Vase Biol. 26:1094-1100, 2006; Cohen J et al. Nat. Genet. 37:161-165,

2005; Horton J et al. Trends Biochem Sci. 32 (2):71-77, 2009).

[0004] Hyperlipidemia is a major risk factor for the atherosclerotic cardiovascular disease, 60%

of coronary atherosclerotic heart disease and 40% of ischemic stroke are caused by a prolonged

elevation of low-density lipoprotein cholesterol (LDL-C) (Stone NJ et al. J Am Coll Cardiol.

63: 2889-2934, 2014). Statins are still the main force in lowering LDL-C clinically at present,

but the LDL-C of the 60-70% patients administered with statins still fails to reach the desirable level (Barkas F et al. Angiology. 66:346-353, 2015; Krahenhuhl S et al. Drugs. 76:1175-1190,

2016). In view of the significant effect of inhibiting PCSK9 in reducing LDL-C and the

incidence of coronary heart disease, several therapeutic regimens are under development of

drugs that block PCSK9 in order to reduce LDL-C and the incidence of coronary heart disease.

Anti-PCSK9 monoclonal antibodies can effectively prevent PCSK9 from binding to LDL-R,

allowing more LDL-R to be expressed by the liver, thereby lowering LDL-C level of plasma

(Mullard A. Nat Rev Drug Discov. 11:817-819, 2012; Rosenson RS et al. J Am Coll Cardiol.

72:314-329, 2018). PCSK9 antibodies are currently clinically administered by 1-2 injections,

typically 1 month (Zhang XL et al. BMC Med. 13:123-123, 2015), can effectively reduce the

LDL-C.

[0005] Small interfering RNA (siRNA) molecules can reduce PCSK9 level (Fizgerald K et al. Lancet. 383:60-68, 2014). The siRNA molecules utilize the intracellular naturally occurring

RNA interference (RNAi) pathway, bind to the intracellular RNA Induced Silencing Complex

(RISC) to specifically cleave the messenger RNA (mRNA) molecules encoding PCSK 9. The

cleaved mRNA will be degraded and cannot be translated into protein, which in turn results in

the reduced protein level of PCSK 9. Therefore, it has important significance to study effective

siRNA molecules to inhibit PCSK9 and thereby performing treatment for the related diseases.

SUMMARY

[0006] The present disclosure aims to overcome the problems in the prior art, and provides a

novel nucleic acid targeted to PCSK9 and uses thereof.

[0007] A first aspect of the present disclosure provides a nucleic acid comprising a sense

strand and an antisense strand, wherein the sense strand contains a sequence having at least 80%

sequence identity with a sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,

SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,

SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID

NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ

ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,

SEQ ID NO:53, SEQ ID NO:55 or SEQ ID NO:57; the antisense strand contains a sequence

having at least 80% sequence identity with a sequence set forth in SEQ ID NO:2, SEQ ID NO:4,

SEQ IDNO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO: 58.

[0008] A second aspect of the present disclosure provides a targeted drug delivery system comprising a target group, a linkage group, and the aforementioned nucleic acid connected with the target group via the linkage group.

[0009] A third aspect of the present disclosure provides a pharmaceutical composition comprising the aforementioned nucleic acid or targeted drug delivery system and a pharmaceutically acceptable carrier.

[0010] A fourth aspect of the present disclosure provides an use of the aforementioned nucleic acid, targeted drug delivery system or pharmaceutical composition in manufacture a medicament for treating and/or preventing hyperlipidemia related diseases.

[0011] A fifth aspect of the present disclosure provides an use of the aforementioned nucleic acid, targeted drug delivery system or pharmaceutical composition in manufacture a medicament for reducing the expression level of proprotein convertase subtilisin/kexin 9.

[0012] The nucleic acids of the present disclosure are capable of effectively reducing the PCSK9 levels, have a stronger safety and longer half-life than antibodies, and are not prone to cause drug immunogenicity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 illustrates an effect of siRNA in reducing serum PCSK9 protein in humanized PCSK9 mice;

[0014] FIG. 2 illustrates an effect of siRNA in reducing liver PCSK9 mRNA expression in humanized PCSK9 mice;

[0015] FIG. 3 illustrates an effect of siRNA dose-dependently decreasing serum PCSK9 concentration of humanized PCSK9 mice.

DETAILED DESCRIPTION

[0016] The terminals and any value of the ranges disclosed herein are not limited to the precise

ranges or values, such ranges or values shall be comprehended as comprising the values

adjacent to the ranges or values. As for numerical ranges, the endpoint values of the various

ranges, the endpoint values and the individual point value of the various ranges, and the

individual point values may be combined with one another to produce one or more new

numerical ranges, which should be deemed have been specifically disclosed herein.

[0017] The present disclosure provides a (modified or unmodified) nucleic acid comprising a sense strand and an antisense strand, the sense strand contains a sequence having at least 80%

SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID

ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,

SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,

SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID

NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,

SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO: 58.

[0018] Wherein a sequence having at least 80% sequence identity refers to the circumstance

of including 0, 1, 2, 3 or 4 different bases with the referred sequence, as well as the circumstance

of additionally binding with more bases on the basis of 0, 1, 2, 3 or 4 different bases with the

referred sequence. Furthermore, the different bases may be located anywhere in the referred

sequence, but preferably, along the 5'-3' direction, the different bases in the sense strand are the

last 1-4 bases, and the different bases in the antisense strand are the first 1-4 bases.

[0019] More preferably, the sense strand has 16-30 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) nucleotides (bases). More preferably, the antisense strand has 16-30 (e.g.,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) nucleotides (bases).

[0020] In the present disclosure, the sense strand and the antisense strand may have the same or different length.

[0021] All the nucleotide acid groups in the aforesaid nucleic acid may be chemically unmodified, or contain at least one modified nucleotide group, and the modification may be on a random site of the nucleotide.

[0022] In preferred embodiments of the present disclosure, as shown in Table 1, the nucleic acid is at least one selected from siRNA-1 having a sense strand sequence of SEQ ID NO:1 and an antisense strand sequence of SEQ ID NO:2, siRNA-2 having a sense strand sequence of SEQ ID NO:3 and an antisense strand sequence of SEQ ID NO:4, siRNA-3 having a sense strand sequence of SEQ ID NO:5 and an antisense strand sequence of SEQ ID NO:6, siRNA-4 having a sense strand sequence of SEQ ID NO:7 and an antisense strand sequence of SEQ ID NO:8, siRNA-5 having a sense strand sequence of SEQ ID NO:9 and an antisense strand sequence of SEQ ID NO:10, siRNA-6 having a sense strand sequence of SEQ ID NO:11 and an antisense strand sequence of SEQ ID NO:12, siRNA-7 having a sense strand sequence of SEQ ID NO:13 and an antisense strand sequence of SEQ ID NO:14, siRNA-8 having a sense strand sequence of SEQ ID NO:15 and an antisense strand sequence of SEQ ID NO:16, siRNA-9 having a sense strand sequence of SEQ ID NO:17 and an antisense strand sequence of SEQ ID NO:18, siRNA 10 having a sense strand sequence of SEQ ID NO:19 and an antisense strand sequence of SEQ ID NO:20, siRNA-11 having a sense strand sequence of SEQ ID NO:21 and an antisense strand sequence of SEQ ID NO:22, siRNA-12 having a sense strand sequence of SEQ ID NO:23 and an antisense strand sequence of SEQ ID NO:24, siRNA-13 having a sense strand sequence of SEQ ID NO:25 and an antisense strand sequence of SEQ ID NO:26, siRNA-14 having a sense strand sequence of SEQ ID NO:27 and an antisense strand sequence of SEQ ID NO:28, siRNA 15 having a sense strand sequence of SEQ ID NO:29 and an antisense strand sequence of SEQ ID NO:30, siRNA-16 having a sense strand sequence of SEQ ID NO:31 and an antisense strand sequence of SEQ ID NO:32, siRNA-17 having a sense strand sequence of SEQ ID NO:33 and an antisense strand sequence of SEQ ID NO:34, siRNA-18 having a sense strand sequence of SEQ ID NO:35 and an antisense strand sequence of SEQ ID NO:36, siRNA-19 having a sense strand sequence of SEQ ID NO:37 and an antisense strand sequence of SEQ ID NO:38, siRNA 20 having a sense strand sequence of SEQ ID NO:39 and an antisense strand sequence of SEQ

ID NO:40, siRNA-21 having a sense strand sequence of SEQ ID NO:41 and an antisense strand

sequence of SEQ ID NO:42, siRNA-22 having a sense strand sequence of SEQ ID NO:43 and

an antisense strand sequence of SEQ ID NO:44, siRNA-23 having a sense strand sequence of

SEQ ID NO:45 and an antisense strand sequence of SEQ ID NO:46, siRNA-24 having a sense

strand sequence of SEQ ID NO:47 and an antisense strand sequence of SEQ ID NO:48, siRNA

25 having a sense strand sequence of SEQ ID NO:49 and an antisense strand sequence of SEQ

ID NO:50, siRNA-26 having a sense strand sequence of SEQ ID NO:51 and an antisense strand

sequence of SEQ ID NO:52, siRNA-27 having a sense strand sequence of SEQ ID NO:53 and

an antisense strand sequence of SEQ ID NO:54, siRNA-28 having a sense strand sequence of

SEQ ID NO:55 and an antisense strand sequence of SEQ ID NO:56, siRNA-29 having a sense

strand sequence of SEQ ID NO:57 and an antisense strand sequence of SEQ ID NO: 58.

[0023] The nucleic acid according to the present disclosure, wherein the nucleic acid comprises a nucleotide group as the basic structural unit, the nucleotide group comprising a

phosphate group, a ribose group and a base group, preferably the nucleic acid comprises at least

one modified nucleotide group. The modification will not result in the functional loss of the

nucleic acid inhibiting the PCSK9, or the modified nucleic acid has an efficiency of inhibiting

PCSK9 not less than 50% (e.g., 50%, 51%, 52%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,

63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98% or 99%) of the nucleic acid before modification.

[0024] The nucleic acid according to the present disclosure, wherein the modified nucleotide

group is a nucleotide group in which the phosphate group and/or the ribose group is modified.

The site with a modification may be at least the site 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, 30 of the nucleotides at the site 1, 2, 3,

4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30of

the sense strand and/or the antisense strand.

[0025] For example, modification of the phosphate group refers to the modification of oxygen

in the phosphate group, including phosphorothioate modification and boranophosphate

modification, etc. The oxygen in the phosphate group is substituted by sulfur, borane, amidogen,

alkyl or alkoxy, respectively, as shown in the formulae below. Each of the modifications can stabilize structure of the nucleic acid, maintain high specificity and high affinity for the base pairing.

Base Base

O OH O OH S-P=O H-B-P=O OH 1 Base Base _0 0

O OH O OH

Phosphorthioate modification Boranophosphate modification

0O Base S Base

OR Si=X 0 as - 0R OR=

Base Base

O R O R

Non-stereospecificphosphorothioate Stereospecific(Sp)phosphorothioate modification modification

O) Base O Base

O R R' O R H 2 C-P=X

Base O Base

O R O R

Stereospecific (Rp) phosphorothioate modification Alkyl phosphate modification

U WBase O U0 ~Base

R" O R H O R O-P=X N-P=X

> ~Base 00 yBase

O R ,OR

Alkoxyl phosphate modification Phosphoramidate modification

u ~ Base

NH R H. I N-P=X H I 0 ~Base

O R

Phosphorodiamidate modification

[0026] In the above structural formula, Base denotes the base A, U, C, G or T. X may be

oxygen (0) or sulfur (S). R may be the same or different in the above structure, such as hydrogen

(H), fluorine (F), methoxyl (OME) or Methoxyethyl (MOE), hydroxyl, allyl, ethylamino,

propargyl, amino, cyanoethyl, acetyl etc.; R' and R" may each independently be hydrogen (H),

methyl (CH 3), ethyl (CH 2 CH3), propyl (CH 2CH 2CH 3), isopropyl (CH(CH 3)2 ), allyl, propargyl,

acyloxybenzyl and acyloxyethyl.

[0027] Modification of the ribose group refers to modification of the 2'-hydroxyl group (2'

OH) in the ribose group. After introducing certain substituents (e.g., methoxyl or fluorine) at

the 2' -hydroxyl of the ribose group, the nucleic acid is not readily cleaved by ribonuclease,

thereby enhancing stability of the nucleic acid and allowing the nucleic acid to be more resistant

to hydrolysis by the nuclease. Modifications of the 2'-hydroxyl group in the nucleotide pentose

include 2'-fluoro modification (e.g., 2'-arabino-fluoro modification), 2'-methoxy modification

(2'-OME), 2'-methoxyethyl modification (2'-MOE), 2'-2,4-dinitrophenol modification (2'

DNP modification), 2',4'-constrained ethyl modification, 2'-Amino modification, 2'-Deoxy modification, BNA, acyclic nucleic acid modification, mal-positioned nucleic acid modification, L-type nucleic acid modification and the like. BNA (internal ring bridged nucleotide) refers to a constrained or inaccessible nucleotide. BNA can contain a bridging structure of a five-, six-, or seven-membered ring. The bridge is typically incorporated into the

2'-, 4'-position of the ribose ring to provide the 2',4'-BNA nucleotides such as locked ethyl

modification (LNA), constrained ethyl modification (ENA), and constrained ethyl bicyclic

nucleic acid modification (cET BNA). Acyclic nucleic acid is a nucleotide formed after the

ribose ring of the nucleotide is opened, such as unlocked nucleic acid (UNA) nucleotides and

glycerol nucleic acid (GNA) nucleotides. Mal-positioned nucleic acid modification refers to a

3',5'-phosphate bond linkage substituted by the 2',5'-phosphate bond linkage. L-type nucleic

acid modification refers to a naturally occurring D-type nucleic acid being substituted by its

mirror-stereoscopic counterpart L-type nucleic acid.

-O -0 Base Base o 0

O F 0 O-CH 3

2'-fluoro modification 2'-OME modification

-0 -0 Base Base o _0 H 2 H2 O -C -C O-CH 3 O DNP

2'-MOE modification 2'-DNP modification

-0 -0 _0 Base Base Base ')~BasBa0e

0 O NH 2 O H 0 .

O-P=O

Locked nucleic acid modification 2'-amino modification 2'-Deoxy modification

-O Base O Base o 0

O O O-P=O O-P=O o 0 ENA modification cET BNA modification

-O Base O Base

O R O R

Unlocked nucleic acid modification Glycerol nucleic acid modification

Base Base O -O_

R 0 R Oy

Mal-positioned nucleic acid modification L-type nucleic acid modification

[0028] In the above structural formula, Base denotes the base A, U, C, G or T. R may be the

same or different in the above structure, such as hydrogen (H), fluorine (F), methoxyl (OME)

or Methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl and acetyl.

[0029] The nucleic acid according to the present disclosure, wherein the nucleotide group in

which the ribose group is modified is preferably the nucleotide group in which the 2'-OH of the

ribose group is substituted by methoxyl or fluorine.

[0030] According to a particularly preferred embodiment of the present disclosure, wherein

the sense strand of nucleic acid comprising the nucleotide group of uracil base or cytosine base,

which is the nucleotide group in which the ribose group is modified, that is, the 2'-OH of the

ribose group in the sense strand of nucleic acid comprising the nucleotide group of uracil base

or cytosine base is substituted by methoxyl or fluorine. More preferably, the 3'-end of both the sense strand and antisense strand of nucleic acid may be linked with dTdT; alternatively, the 3' end of the antisense strand of nucleic acid may be linked with AA or UU, or any combination of two nucleic acids (the nucleic acids may be but are not limited to CC, GG or UG), to provide the sequence with specificity as inducement to mRNA degradation. Nucleic acids with such modifications exhibit more excellent in vivo inhibitory effect, and said modifications may further reduce the in vivo immunogenicity of the nucleic acids of the present disclosure.

[0031] The nucleic acid of the present disclosure may further comprise the modification in which a monophosphate nucleoside is linked to the 5'-end of the antisense strand. The 5'

monophosphate at the end of the guide strand of the siRNA is important for RISC recognition.

Wherein phosphorylation of the 5'-hydroxyl group plays a certain role on whether the siRNA

can be effectively loaded on the intracellular Ago2. The monophosphate at 5'-end of the guide

strand in the siRNA has interaction with Argonaute-2(Ago2) through the Hydrogen bond, in

order to ensure accurate targeting and precise cleavage of the mRNA target. Several derivatives

of the 5' -monophosphate nucleosides are commonly used, this type of derivative of the

phosphate nucleoside has been proven to exhibit certain stability in the biological metabolism

medium, and to play a certain role in facilitating the loading of siRNA guide strand on the

intracellular Ago2 (Nucleic Acids Research, 2015, 43, 2993-3011). The nucleic acid according

to the present disclosure, wherein the trans-vinyl phosphate (VP) is preferably the first choice,

the nucleic acid may comprise derivatives of the monophosphate nucleoside other than those

mentioned above.

PO 0-O 00

0 Base Base 0 0

OR 1 O R

5'-monophosphate modification 5'- trans vinyl phosphate modification

- s

Base 0 Base o 0

O R O R

5'-ethylphosphatemodification 5'-phosphorothioatemodification - 0~ 'P

0 IBase

O R

5'- methoxymethylphosphonic acid modification

[0032] In the above structural formula, Base denotes the base A, U, C, G or T. R may be the same or different in the above structure, such as hydrogen (H), fluorine (F), methoxyl (OME)

or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino and acetyl.

[0033] According to the preferred embodiments of the present disclosure, the base sequences

of the modified nucleic acids and the modification modes are as shown in Table 2 (f, s,

underlined), i.e., the sense strand and antisense strand of the modified nucleic acids siRNA-9,

siRNA-13, siRNA-16, siRNA-19 and siRNA-23 have the modifications indicated by f, s and

underline as shown in Table 2, and the 3' end of the antisense strand is further connected with

UU.

[0034] In a particularly preferred embodiment, the nucleic acid is selected from siRNA-23

having a sense strand sequence of SEQ ID NO: 45 and an antisense strand sequence of SEQ ID

NO: 46, and having the modifications at the nucleotide as follows:

[0035] 5'-GsCsCAAGCfCUfCUfUCUUACUUCA -3'

[0036] 5'-UsGfsAAfGUfAAGAAGAGfGCfUUGGCsUsU-3'

[0037] wherein the lowercase letter f indicates that the nucleotide adjacent to the left side of

the letter f is a 2'-fluoro modified nucleotide (i.e., the 2'-OH of the pentose in the nucleotide is

substituted by fluorine); the lowercase letter s indicates that two nucleotides adjacent to the left side and right side of the letter s are connected through a thiophosphate diester bond (i.e., the non-bridging oxygen atom in the phosphodiester bond is substituted by sulfur atom); the underlined nucleotide indicates that the 2' hydroxyl group of the nucleotide is substituted by methoxyl group. Such modifications result in higher in vivo activity of the nucleic acid (siRNA).

[0038] The nucleic acid according to the present disclosure can be obtained by conventional methods in the art, for example through the solid-phase synthesis and solution-phase synthesis, wherein the solid-phase synthesis has the commercial customization service, thus it is commercially available. The modified nucleotide group can be introduced by means of the nucleotide monomer having the corresponding modification.

[0039] Based on the synthesized nucleic acid (siRNA) mentioned above, the present disclosure can further construct an expression plasmid of the shRNA having the identical or similar function with the siRNA, and the method for constructing the expression plasmid is well-known among those skilled in the art, the content will not be described in detail herein.

[0040] The present disclosure further provides a target gene sequence of a nucleic acid as described above. In some embodiments, the target gene sequence is set forth in any item of column 2 of Table 1.

[0041] Table 1

No. Target Sense strand sequence (5'-3') Antisense strand sequence (5'-3') gene sites 1 3317-3337 AGCUCGGUGAGUGAUGGCAGA(1) UCUGCCAUCACUCACCGAGCU (2) 2 3318-3336 GCUCGGUGAGUGAUGGCAG(3) CUGCCAUCACUCACCGAGC(4) 3 3183-3201 GCUCACACAGCAGGAACUG (5) CAGUUCCUGCUGUGUGAGC (6) 4 2117-2135 AGUCAAGGAGCAUGGAAUC (7) GAUUCCAUGCUCCUUGACU (8) 5 2730-2748 UCCUCAGGUCUCCACCAAG (9) CUUGGUGGAGACCUGAGGA (10) 6 2890-2908 UGGAGGCUUAGCUUUCUGG (11) CCAGAAAGCUAAGCCUCCA(12) 7 3199-3217 CUGAGCCAGAAACGCAGAU (13) AUCUGCGUUUCUGGCUCAG (14) 8 1889-1907 CUGCAGCGUCCACACAGCU (15) AGCUGUGUGGACGCUGCAG (16) 9 3318-3338 GCUCGGUGAGUGAUGGCAGAA (17) UUCUGCCAUCACUCACCGAGC(18) 10 626-644 GCAUGUCUUCCAUGGCCUU (19) AAGGCCAUGGAAGACAUGC(20) 11 827-845 GGUGGAGGUGUAUCUCCUA(21) UAGGAGAUACACCUCCACC (22) 12 893-911 GGUCACCGACUUCGAGAAU (23) AUUCUCGAAGUCGGUGACC(24) 13 893-913 GGUCACCGACUUCGAGAAUGU (25) ACAUUCUCGAAGUCGGUGACC(26) 14 3039-3059 AGCCAUCACCUAGGACUGACU (27) AGUCAGUCCUAGGUGAUGGCU(28) 15 2612-2632 GCAUUUCACCAUUCAAACAGG (29) CCUGUUUGAAUGGUGAAAUGC(30) 16 3466-3484 CCCAAGCAAGCAGACAUUU (31) AAAUGUCUGCUUGCUUGGG (32)

17 3433-3451 CCAACAACUGUCCCUCCUU (33) AAGGAGGGACAGUUGUUGG (34) 18 3399-3418 GGAGAUGCUUCUAAGGCAU (35) AUGCCUUAGAAGCAUCUCC (36) 19 3192-3210 GCAGGAACUGAGCCAGAAA (37) UUUCUGGCUCAGUUCCUGC (38) 20 2900-2918 GCUUUCUGGAUGGCAUCUA (39) UAGAUGCCAUCCAGAAAGC (40) 21 2880-2898 CCCUGAUUAAUGGAGGCUU (41) AAGCCUCCAUUAAUCAGGG (42) 22 2744-2762 CCAAGGAGGCAGGAUUCUU (43) AAGAAUCCUGCCUCCUUGG (44) 23 3233-3253 GCCAAGCCUCUUCUUACUUCA (45) UGAAGUAAGAAGAGGCUUGGC(46) 24 2969-2989 GGGCAUUUCACCAUUCAAACA (47) AGUAAAGGUGGCUCAGGUUUC(48) 25 2978-2998 GCCACCUUUACUCUGCUCUAU (49) AUAGAGCAGAGUAAAGGUGGC(50) 26 3471-3489 GCAAGCAGACAUUUAUCUU (51) AAGAUAAAUGUCUGCUUGC(52) 27 2610-2628 GGGCAUUUCACCAUUCAAA (53) UUUGAAUGGUGAAAUGCCC(54) 28 2612-2630 GCAUUUCACCAUUCAAACA (55) UGUUUGAAUGGUGAAAUGC(56) 29 2722-2740 GCAUUCAAUCCUCAGGUCU (57) AGACCUGAGGAUUGAAUGC(58)

Note: "(1)" recited in columns 3-4 denotes SEQ ID NO:1, the number "3317-3337" recited in column 2

represents nucleotides at sites 3317-3337 in PCSK9 gene sequence, and the like.

[0042] The coding sequence of human PCSK9 (NM_174936.4, SEQ ID NO: 59): 1 agcgacgtcg aggcgctcat ggttgcaggc gggcgccgcc gttcagttca gggtctgagc 61 ctggaggagt gagccaggca gtgagactgg ctcgggcggg ccgggacgcg tcgttgcagc 121 agcggctccc agctcccagc caggattccg cgcgcccctt cacgcgccct gctcctgaac 181 ttcagctcct gcacagtcct ccccaccgca aggctcaagg cgccgccggc gtggaccgcg 241 cacggcctct aggtctcctc gccaggacag caacctctcc cctggccctc atgggcaccg 301 tcagctccag gcggtcctgg tggccgctgc cactgctgct gctgctgctg ctgctcctgg 361 gtcccgcggg cgcccgtgcg caggaggacg aggacggcga ctacgaggag ctggtgctag 421 ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc acagccacct 481 tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg gtgctgaagg 541 aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc caggctgccc 601 gccggggata cctcaccaag atcctgcatg tcttccatgg ccttcttcct ggcttcctgg 661 tgaagatgag tggcgacctg ctggagctgg ccttgaagtt gccccatgtc gactacatcg 721 aggaggactc ctctgtcttt gcccagagca tcccgtggaa cctggagcgg attacccctc 781 cacggtaccg ggcggatgaa taccagcccc ccgacggagg cagcctggtg gaggtgtatc 841 tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc atggtcaccg 901 acttcgagaa tgtgcccgag gaggacggga cccgcttcca cagacaggcc agcaagtgtg 961 acagtcatgg cacccacctg gcaggggtgg tcagcggccg ggatgccggc gtggccaagg 1021 gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg gttagcggca 1081 ccctcatagg cctggagttt attcggaaaa gccagctggt ccagcctgtg gggccactgg 1141 tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct caacgccgcc tgccagcgcc 1201 tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa cttccgggac gatgcctgcc 1261 tctactcccc agcctcagct cccgaggtca tcacagttgg ggccaccaat gcccaagacc 1321 agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac ctctttgccc 1381 caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg tcacagagtg 1441 ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg tctgccgagc 1501 cggagctcac cctggccgag ttgaggcaga gactgatcca cttctctgcc aaagatgtca 1561 tcaatgaggc ctggttccct gaggaccagc gggtactgac ccccaacctg gtggccgccc

1621 tgccccccag cacccatggg gcaggttggc agctgttttg caggactgta tggtcagcac 1681 actcggggcc tacacggatg gccacagccg tcgcccgctg cgccccagat gaggagctgc 1741 tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg gaggcccaag 1801 ggggcaagct ggtctgccgg gcccacaacg cttttggggg tgagggtgtc tacgccattg 1861 ccaggtgctg cctgctaccc caggccaact gcagcgtcca cacagctcca ccagctgagg 1921 ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca ggctgcagct 1981 cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg ccacgaggtc 2041 agcccaacca gtgcgtgggc cacagggagg ccagcatcca cgcttcctgc tgccatgccc 2101 caggtctgga atgcaaagtc aaggagcatg gaatcccggc ccctcaggag caggtgaccg 2161 tggcctgcga ggagggctgg accctgactg gctgcagtgc cctccctggg acctcccacg 2221 tcctgggggc ctacgccgta gacaacacgt gtgtagtcag gagccgggac gtcagcacta 2281 caggcagcac cagcgaaggg gccgtgacag ccgttgccat ctgctgccgg agccggcacc 2341 tggcgcaggc ctcccaggag ctccagtgac agccccatcc caggatgggt gtctggggag 2401 ggtcaagggc tggggctgag ctttaaaatg gttccgactt gtccctctct cagccctcca 2461 tggcctggca cgaggggatg gggatgcttc cgcctttccg gggctgctgg cctggccctt 2521 gagtggggca gcctccttgc ctggaactca ctcactctgg gtgcctcctc cccaggtgga 2581 ggtgccagga agctccctcc ctcactgtgg ggcatttcac cattcaaaca ggtcgagctg 2641 tgctcgggtg ctgccagctg ctcccaatgt gccgatgtcc gtgggcagaa tgacttttat 2701 tgagctcttg ttccgtgcca ggcattcaat cctcaggtct ccaccaagga ggcaggattc 2761 ttcccatgga taggggaggg ggcggtaggg gctgcaggga caaacatcgt tggggggtga 2821 gtgtgaaagg tgctgatggc cctcatctcc agctaactgt ggagaagccc ctgggggctc 2881 cctgattaat ggaggcttag ctttctggat ggcatctagc cagaggctgg agacaggtgc 2941 gcccctggtg gtcacaggct gtgccttggt ttcctgagcc acctttactc tgctctatgc 3001 caggctgtgc tagcaacacc caaaggtggc ctgcggggag ccatcaccta ggactgactc 3061 ggcagtgtgc agtggtgcat gcactgtctc agccaacccg ctccactacc cggcagggta 3121 cacattcgca cccctacttc acagaggaag aaacctggaa ccagaggggg cgtgcctgcc 3181 aagctcacac agcaggaact gagccagaaa cgcagattgg gctggctctg aagccaagcc 3241 tcttcttact tcacccggct gggctcctca tttttacggg taacagtgag gctgggaagg 3301 ggaacacaga ccaggaagct cggtgagtga tggcagaacg atgcctgcag gcatggaact 3361 ttttccgtta tcacccaggc ctgattcact ggcctggcgg agatgcttct aaggcatggt 3421 cgggggagag ggccaacaac tgtccctcct tgagcaccag ccccacccaa gcaagcagac 3481 atttatcttt tgggtctgtc ctctctgttg cctttttaca gccaactttt ctagacctgt 3541 tttgcttttg taacttgaag atatttattc tgggttttgt agcattttta ttaatatggt 3601 gactttttaa aataaaaaca aacaaacgtt gtcctaa

[0043] The present disclosure also provides a targeted drug delivery system comprising a

target group, a linkage group, and the aforementioned nucleic acid connected with the targeted

group via the linkage group. Wherein the targeted group is capable of further improving

targeting performance of the small nucleic acid, it may be provided by a monosaccharide (e.g.,

glucose, mannose, allose, altrose, galactose, galactosamine, N-acetylgalactosamine, talose,

fructose, idose) and/or a polypeptide (e.g., protein, monoclonal antibody, nanoparticle). The

linkage group may be selected from -O-[CH 2 CH20]-, -[CH 2]m-CONH-[CH 2 ]nO-, -0

[CH 2CH 20]m-CONH-[CH 2 ]nO-, -0-[CH 2 ]m-CONH- [CH 2 H2 0]nO-. Wherein m and n each may

independently be an integer from 1 to 10.

[0044] According to a preferred embodiment of the present disclosure, the targeted drug delivery system has a structure shown below, wherein Nu represents a nucleic acid (siRNA) of

the present disclosure, wherein the compound moiety can be coupled with the 5' end or the 3'

end of the sense strand of siRNA via a phosphodiester bond, or can be coupled with the 5' end

or the 3' end of the antisense strand of siRNA via a phosphodiester bond. Specifically, the

compound moiety can be synthesized with a nucleoside monomer or a nucleoside attached to a

solid phase carrier under coupling reaction conditions and in the presence of a coupling reagent,

thereby allowing the compound moiety to be attached to the nucleic acid via coupling reaction.

The targeted drug delivery system can improve the cell penetrating capability of the nucleic

acid drug (Nu) by using the structural characteristics on the left side thereof, thereby enhance

the stability of Nu in cells, and the preparation process is simple and highly practical. OH OH

N NHAc H

HO N N H NHAc H N O H 0 OH OH 0

HO O O O N

[0045] NHAc H

Formula (603)

[0046] The present disclosure further provides a pharmaceutical composition comprising the

aforementioned nucleic acid or targeted drug delivery system and a pharmaceutically

acceptable carrier. The pharmaceutical composition can be prepared with the nucleic acid and

the pharmaceutically acceptable carrier through conventional method. For example, the

pharmaceutical composition may be an injection solution. The injection solution can be used

for subcutaneous, intramuscular or intravenous injection.

[0047] The pharmaceutical composition according to the present disclosure, wherein the

dosage of nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable

carrier are not particularly defined, typically the amount of the pharmaceutically acceptable

carrier may be within a range of 1-100,000 parts by weight (e.g. 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1,000 parts by weight, 5,000 parts by weight, 10,000 parts by weight, 50,000 parts by weight, 100,000 parts by weight or a random value between any two numerical values mentioned above) relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated in terms of the nucleic acid).

[0048] The pharmaceutical composition according to the present disclosure, wherein the pharmaceutically acceptable carrier may be various carrier conventionally used in the art, for

example, the pharmaceutically acceptable carrier may include at least one of a pH buffer

solution, a protective agent and an osmotic pressure conditioning agent. The pH buffer solution

may be a tri(hydroxymethyl)aminomethane hydrochloride buffer having a pH of 7.5-8.5 and/or

a phosphate buffer having a pH of 5.5-8.5, preferably a phosphate buffer having a pH of 5.5

8.5. The protective agent may be at least one of inositol, sorbitol and sucrose. The protective

agent may be contained in an amount of 0.01-30 wt.% (e.g., 0.01 wt.%, 0.05 wt.%, 0.1 wt.%,

0.5 wt.%, 1 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, 30 wt.% or a random value

between any two numerical values thereof) based on the total weight of the pharmaceutical

composition. The osmotic pressure conditioning agent may be sodium chloride and/or

potassium chloride. The osmotic pressure conditioning agent is contained in an amount such

that the osmotic pressure of the pharmaceutical composition is within a range of 200-700

milliosmols per kilogram. The amount of the osmotic pressure conditioning agent may be

determined by those skilled in the art based on the desired osmotic pressure.

[0049] According to a preferred embodiment of the present disclosure, the pharmaceutically

acceptable carrier is a liposome. The liposome may be any liposome capable of encapsulating

nucleic acid, it may have a diameter of 25-1,000nm, and may include but not limited to the

cholesterol and analog or derivative thereof.

[0050] The dosage of the pharmaceutical composition according to the present disclosure may

be a conventional dosage in the art, the dosage may be determined according to various

parameters, in particular the age, body weight and sex of a subject. For example, for female

mice having a weight of 25-30g at an age of 3-4 months, the pharmaceutical composition may

be used in a dosage of 0.01-100mg/kg body weight, preferably 1-10mg/kg body weight, based

on the amount of nucleic acid in the pharmaceutical composition.

[0051] The present disclosure also provides an use of the aforementioned nucleic acid, targeted drug delivery system or pharmaceutical composition in manufacture a medicament for

treating and/or preventing hyperlipidemia related diseases. In the pharmaceutical composition

for treating and/or preventing hyperlipidemia related diseases, the nucleic acid functions mainly

through the mechanism of RNA interference.

[0052] The present disclosure further provides an use of the aforementioned nucleic acid, targeted drug delivery system or pharmaceutical composition in manufacture a medicament for

reducing the expression level of proprotein convertase subtilisin/kexin 9 (e.g., in serum or liver).

[0053] The present disclosure also provides a method of inhibiting PCSK9, the method comprises administering the nucleic acid and/or pharmaceutical composition to a patient

suffering from the hyperlipidemia related diseases. "inhibiting" refers to silencing the gene

expression of PCSK 9. The patient may be a mammal, preferably a primate, more preferably a

human. Administration may be performed through various routes, depending on whether the

local or systemic treatment is required. The administration mode may be, but is not limited to,

intravenous administration, intraarterial administration, subcutaneous administration, intraperitoneal administration, transdermal administration (e.g., by implantation of a device),

and intra soft tissue administration.

[0054] "Hyperlipidemia related diseases" may include coronary atherosclerosis heart disease,

ischemic stroke, arteriosclerosis cerebral infarction, hypertension, diabetes mellitus, nephropathy (or kidney disease), peripheral vascular diseases and the like.

[0055] In addition, the present disclosure further provides a method of inhibiting PCSK9 in

vitro, the method comprises introducing the nucleic acid and/or pharmaceutical composition

into a cell.

[0056] The present disclosure will describe in detail below with reference to examples. Unless

otherwise specified in the present disclosure, the reagents and culture media used therein are

commercially available, the nucleic acid electrophoresis and other operations used in the

present disclosure is performed in a conventional manner.

[0057] Example 1

[0058] The siRNA-1 to siRNA-29 listed in Table 1 were obtained by a solid phase synthesis

method. 0.5 ml of cell culture fluid (DMEM, 10% FBS) containing 105 Hep3B cells was added into a 24-well cell culture dish, and cultured overnight in a cell culture vessel containing 5%

CO2 at the temperature of 37°C. Lipofectamine@ RNAiMAX (1-2 microliters/well) and small

interfering nucleic acid siRNA-1 to siRNA-29 as shown in Table 1 were added into the serum

free cell culture fluid, and the serum-free cell culture fluid was added into the cell culture wells

such that the final concentration of small interfering nucleic acid per well was 33nM or100nM,

and continued to culture for 48 hours in the cell culture vessel containing 5% C02 at the

temperature of 37°C. In order to extract RNA, the cell culture supernatant was pipetted

completely, washed with PBS, and after pipetting completely, the RNA was extracted according

to the operating instruction of the RNAeasy Mini kit (QIAGEN, Article No. 74104). RT-PCR

was performed according to the recommendation of High Capacity cDNA Reverse

Transcription Kits (Thermo Fisher, Article No. 4368814), and 0.5 or 1 g of RNAwas contained

in each reaction. The gene expression was quantified by using the real-time fluorescence PCR

method, and the TaqMan probe of human PCSK9 was Hs00545399_ml, and the probe of the

internal reference gene (human HPRT1) was Hs02800695_ml (Thermo Fisher Scientific,

Waltham, MA, USA). The PCR conditions were 1 cycle for 20 sec at the temperature of 95°C,

40 cycles for 1 see at 95°C and 20 see at 60°C, and the real-time fluorescence PCR instrument

was StepOne Plus (Thermo Fisher). The PCSK9 gene expression was calculated based on the

2A-AACt, and the human HPRT1 gene expression was used as the internal reference. The

expression level of PCSK9 gene was expressed as a percentage of treated group vs. control

group (using RNAiMAX only) (see Table 2).

[0059] Table 2 No. 33 nM (%) 1OOnM ( %) 1 38.9 32.0 2 43.5 59.1 3 28.1 25.7 4 22.4 21.6 5 55.4 56.2 6 37.6 62.9 7 39.8 59.8 8 85.1 111.7 9 34.1 31.2 10 27.7 30.5 11 73.4 70.7

12 61.7 58.7 13 15.7 11.5 14 18.6 17.4 15 16.6 13.7 16 16.0 12.8 17 54.1 47.8 18 20.7 17.1 19 22.1 16.9 20 18.0 15.8 21 16.4 12.7 22 23.6 12.6 23 22.8 18.4 24 63.5 52.2 25 24.4 16.7 26 23.4 16.2 27 20.6 20.3 28 28.2 17.0 29 33.5 18.6

[0060] Example 2

[0061] (I) Preparation of siRNA drug according to the following steps.

[0062] Synthesis of conjugate (603A)

[0063] 1. Compound 7 was prepared according to the following route: OAc OAc TMSOTf OAc OAc TMSOTf/DCM, rt, OAc OAc

AcO AOAc AcO 0 AcO 0 N3 NHAC CI-CH 2CH 2 -Cl rt l4hrs HN 0 -- '-'N`0c

4 5 6

1) Pd/C, H 2 , EtOAc OAc OAc 6 2) HCI, 2M, 1eq AcO c'--O O O NH3-Cl^ NHAc

7

[0064] Synthesis of compound oxazoline 5

[0065] N-acetylgalactosamine tetraacetate 4 (10g, 25.68 mmol) was dissolved in

dichloroethane (60 mL) at room temperature, trimethylsilyl trifluoromethanesulfonate (8.6g,

38.66 mmol) was added to the aforesaid solution under the stirring condition, stirring was

continued and the solution was heated to 50°C. After reaction at 50°C for 2 hours, the heating

was stopped, and the stirring was continued for 12 hours at room temperature. The solution was

poured into ice water containing saturated sodium bicarbonate, extracted with dichloromethane, and the organic phase was subjected to washing with water. The organic phase was separated out, and dried after adding anhydrous sodium sulfate, and evaporated and dried in reduced pressure to obtain a brownish-yellow foamy syrup-shaped compound 5. The compound 5 was directly used in the next step.

[0066] Synthesis of Compound 6

[0067] The compound oxazoline 5 (4.26g, 12.9 mmol) was dissolved in dichloromethane (20 mL) at room temperature, and mixed with a solution of dry dichloromethane (20 mL) dissolved

with 2-[2-(2-azidoethoxy)ethoxy]ethanol (3.4g, 19 mmol) and stirred under the temperature

condition of 0°C. Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 1.4g, 6.45 mmol) was

added slowly at 0°C into the solution and stirred for 1 hour. The mixed solution was stirred

continuously for 14 hours at room temperature, the solution was then poured into ice water

containing saturated sodium bicarbonate, extracted with dichloromethane (2x50mL), and the

organic phase was subjected to washing with water. The organic phase was separated out, and

dried with anhydrous sodium sulfate, and rotary evaporated and concentrated in reduced

pressure to a semi-dry state. Purification was further performed with a silica gel

chromatographic column, a gradient elution was used, initially rinsed with a mixed solvent

(containing ethyl acetate/methanol, 10:1, v/v), the product components were collected, and the

solvent was drained under reduced pressure to obtain the near-white compound 6 (5.3g, 81%).

H NMR (CDCl3): 6, 6.15 (d, 1H, NH), 5.32 (d, 1H, sugar-H-4'), 5.07 (dd, 1H, J=11.2Hz,

J=3.3Hz, sugar-H-3'), 4.76 (d, 1H, J=8.6Hz, sugar-H-l'),4.17 (m, 3H, sugar-H-2', sugar-H-6'),

3.91 (m, 2H, -CH 20), 3.89 (m, 1H, suger-H-5'), 3.76-3.61 (m, 8H, -CH 20), 3.47 (m, 2H,

CH2N 3), 2.16 (s, 3H, -CH 3, NHAc), 1.99, 2.00, 2.05 (3xs, 9H, -CH3 , Ac). HRMS (ESI) m/z,

C2oH32N4011 (M+H). Theoretical value: 505.49, measured value: 505.20.

[0068] Synthesis of Compound 7

[0069] Azide 6 (522mg, 1.04 mmol) was dissolved in l0mL of ethyl acetate, Pd/C (80mg)

was added into 30mL of ethyl acetate under the protection of nitrogen gas. The reaction bottle

was connected to a hydrogen balloon, subjected to multiple replacements with hydrogen gas,

the reaction bottle was connected to a hydrogen balloon at room temperature, the reaction

solution was continuously stirred for 3 hours. Pd/C was filtered by the Celite, and 0.5mL of

hydrochloric acid (2M) was added dropwise and slowly, and the solution was reacted under continuous stirring for 30 minutes under a reaction temperature of 0°C. 10mL of acetonitrile was added into the reaction solution, and subjected to azeotropic decompression concentration for twice. The concentrated solution was mixed with dichloromethane (10mL), and concentration under reduced pressure was further performed for twice, resulting in an oily foamed crude product 7 (500mg), which was directly used in the next step without further purification. 'H NMR (CDC 3 ): 6, 8.25 (m, 2H, -NH2), 5.34 (d, 1H, sugar-H-4'), 5.21 (dd, 1H,

J=11.2Hz, J=3.3Hz, sugar-H-3'), 4.91 (d, 1H, J=8.5Hz, sugar-H-l'), 4.12 (m, 3H, sugar-H-6',

sugar-H-2'), 4.07 (m, 2H, sugar-H-5', -NH), 3.76 (m, 2H, -CH20), 3.68 (m, 2H, -CH 20), 3.61

(m, 2H, -CH20), 3.58 (m, 4H, 2 x -CH20), 3.20 (m, 2H, NH2), 2.09 (s, 3H, -NHCO 2 CH3), 2.04,

1.96, 1.89 (3 x s, 9H, -CO 2 CH3 ). HRMS (ESI) m/z, C02 H 3 4 N 20 1 1 (M+H+). Theoretical values:

479.49, measured values: 479.20.

[0070] 2. Compound 12 was prepared according to the following route:

NC EtO HO 0 a , 'CN 1) EtOH, H 2SO 4 , 80C HO NH 2 NC O0 NH 2 2 EtO O NH 2 KOH, Dioxane 2)TEAa HO 0 0

8 9 N) 10EtO 0

EtO HO

O e 0 a

(Boc) 20 EtO O N 0 NaOH, 4M HO 0 NH 2 0 H 0 TEA O EtOH, rt, 14hrs O EtO HO 11 12

[0071] Synthesis of compound 9

[0072] Trihydroxymethyl aminomethane 8 (10g, 82.6 mmol) was dissolved in 15 mL of

dioxane, 1.26mL of an aqueous potassium hydroxide solution with a concentration of 40wt%

was dropwise added into the reaction solution and stirred, 20mL of dioxane was further added

at room temperature. Acrylonitrile (18mL, 272 mmol) was added dropwise and slowly to the

reaction flask under the temperature of 0°C, and the entire dropwise adding process was

maintained about 1 hour. The reaction solution was continuously stirred at room temperature

for 24 hours. The reaction solution was poured into a saturated sodium chloride solution, and extracted with dichloromethane (2x5OmL), the organic phase was subjected to washing with water. The organic phase was separated out and dried with the added anhydrous sodium sulfate, and rotary evaporated and concentrated in reduced pressure to a semi-dry state. Purification was further performed with a silica gel chromatographic column, the purified product was first rinsed with dichloromethane and then rinsed with a solvent mixture (containing dichloromethane/methanol, 10:1, v/v), the product components were collected. The product components were subjected to rotary evaporation and concentration in reduced pressure to obtain a pale yellow oily substance 9 (20g, 86%). 'H NMR (CDCl 3): 6, 3.68 (t, 6H, J=7.1Hz, 3 x -CH2 0), 3.44 (s, 6H, 3 x -CH2CNH 2), 2.61 (t, 6H, J=6.2Hz, 3 x -CH2CN), 1.70 (s, 2H, -NH2 ).

HRMS (ESI) m/z, C 13H 20N 4 0 3 (M+H+). Theoretical value: 281.32, measured value: 281.20.

[0073] Synthesis of compound 10

[0074] Tri[(cyanoethoxy)methyl]aminomethane 9 (1.2g, 4.28 mmol) was dissolved in1OmL of anhydrous ethanol, 2mL concentrated sulfuric acid and 1OmL anhydrous ethanol were then

added dropwise and slowly to the solution in a reaction flask at room temperature. The reaction

solution was heated to 80°C and kept at reflux state for about 36 hours. After cooling the

reaction solution to room temperature, 25mL of ice solution of saturated sodium bicarbonate

was added. Ethanol was distilled by rotary evaporation under reduced pressure. The aqueous

solution was extracted with ethyl acetate (2x50mL), the obtained organic phase was dried with

anhydrous sodium sulfate, and then subjected to rotary evaporation and concentration in

reduced pressure to obtain a pale yellow oily substance 10 (0.8g, 46%). This crude product was

directly used in the next reaction without further purification.

[0075] Synthesis of compound 11

[0076] Crude product compound 10 (0.8g, 1.9 mmol) was dissolved in 20mL of

dichloromethane. Di-tert-butyl dicarbonate (2mL, 8.8 mmol) and 5mL of triethylamine were

added to the reaction solution. The reaction solution was stirred for 14 hours at room

temperature. The reaction solution was poured into an aqueous solution containing saturated

sodium bicarbonate, extracted with dichloromethane (2x50mL), and the organic phase was

subjected to washing with water. The organic phase was separated out, and dried with

anhydrous sodium sulfate, and rotary evaporated and concentrated in reduced pressure to a

semi-dry state, purification was further performed with a silica gel chromatographic column, a gradient elution was used, initially washed with dichloromethane solvent, then rinsed with a mixed solvent (containing dichloromethane/methanol, 96:4, v/v), the product components were collected, and the solvent was drained under reduced pressure to obtain a near-white oily substance 11 (0.5g, 51%), 'H NMR (CDC 3): 6, 4.92 (b, 1H, -CONH-), 4.14 (m, 3x2H,

CO 2CH2 -), 3.69 (m, 3x2H, -OCH 2-), 3.63 (s, 3x2H, -OCH2-), 2.53 (m, 3x2H, -COCH 2 -), 1.45 (s, 3x3H, -CH3), 1.26 (t, 3x3H, -CH 2CH3 ). HRMS (ESI) m/z, C 24 H 43 N 1 (M+H+). Theoretical value: 522.60, measured value: 522.40.

[0077] Synthesis of compound 12

[0078] Boc protected compound 11 (0.6g, 1.43 mmol) was dissolved in 20mL of absolute ethanol, 4mL of sodium hydroxide solution (4M) was added dropwise and slowly into the reaction solution, the temperature of reaction solution was kept at 0°C and stirred for 14 hours. The reaction progress was monitored constantly by LC-MS spectrum, while the peak of the reactant was vanished, the reaction solution was subjected to rotary evaporation under reduced pressure, after ethanol was evaporated, lOmL of potassium bisulfate (IM) was added to the reaction solution and continuously stirred for 15 minutes at 0°C. The reaction solution was extracted with ethyl acetate (2x50mL), the obtained organic phase was dried with anhydrous sodium sulfate, then rotary evaporated and concentrated in reduced pressure to obtain a viscous substance 12 (0.5g, 81%). This crude product was directly used in the next step without further purification. 1H NMR (CDCl3): d, 9.40 (b, 3H, -C02HI), 5.0 (b, 1H, -CONH-), 3.70 (m, m, 3x2H, -OCH 2-), 3.65 (s, 3x2H, -OCH2-), 2.60 (m, 3x2H, -COCH 2 -), 1.42 (s, 3x3H, -CH 3 ). HRMS (ESI) m/z, CisH31NO I(M+H+). Theoretical values: 438.32, measured values: 438.20.

[0079] Compound 14 was prepared according to the following route:

HO

AAcCOAc O 0C HO,,,-, N AcO "0_-O--'c rCI + H O NHAc 0 0

7 12 O HO 0 OAc OAc 0 0 AcO O O O N N HAc H OAc OAc HATU, DIPEA 0 0 0 AcO ,'O -O N 0 DCM,/DMF NHAc H N 0 H

OAc OAc 0

AcO NHAc H OAc OAc 13 o0 AcO N NHAc H CAcCOAc 00 0 HCI/DCM AcO O O O 0 0-25°C,lhs, NHAc H NH 2

OAc OAc 0

AcO OO NHAc H 14

[0080] Synthesis of Compound 13

[0081] Tricarboxylic acid 12 (0.5g, 1.14 mmol) was dissolved in 20mL of dichloromethane, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.3g, 3.42 mmol) and

N,N-diisopropylethylamine (0.2g, 3.95 mmol) were added, 8mL of dimethylformamide was

added simultaneously. The compound 7 (2.19g, 4.08 mmol) was dissolved in 5mL of

dimethylformamide, and 1mL of N,N-diisopropylethylamine was added. The two solutions

were blended and stirred at room temperature and continuously stirred for 14 hours. The

complete disappearance of the reactants was confirmed by the chromatographic detection.

20mL of aqueous solution of saturated sodium bicarbonate was added into the reaction solution,

and extracted with 2x5OmL of dichloromethane, the organic phase was rotary evaporated and

concentrated to a semi-dry state, purification was further performed with a silica gel

chromatographic column, a gradient elution was used, initially washed with dichloromethane

solvent, then rinsed with a mixed solvent (containing dichloromethane/methanol, 85:15, v/v),

the product components were collected, the solvent was drained under reduced pressure to obtain the near-yellow oily crude product 13 (2g, 86%). This crude product 13 was further purified by using a reversed-phase chromatographic column, the rinsing solvent was a mixed solvent (containing H20/MeOH, 1:1, v/v), the product components were collected, then rotary evaporated and concentrated to a full dry state to obtain a compound 13 (1.24g, 60%). 'H NMR

(CDC 3): 6, 5.32 (d, 3H, J=3.OHz, sugar-H-4'), 5.18 (dd, 3H, sugar-H-3'), 4.78 (d, 3H, sugar

H-l'), 4.18-4.06 (m, 24H, -OCH 2 , sugar-H-5'), 3.93 (m, 9H, sugar-2xH-6', sugar-H-2'), 3.77,

3.64, 3.46 (m, 6H, -CH2NH-), 2.44, 2.20 (m, 6H, -COCH 2 -), 2.15 (s, 9H, -NHCOCH 3 ), 2.09 (s,

2H, -CH2 -), 2.05, 1.99, 1.95 (3xs, 27H, -OCOCH 3), 1.81 (s, 9H, CH3 , Boc). HRMS (ESI) m/z,

C78H127N7041 (M+2H+)/2. Theoretical value: 910.1, measured value: 910.0.

[0082] Synthesis of Compound 14

[0083] Purified Compound 13 (2g, 1.Immol) was dissolved in 30mL of dichloromethane, 1mL of hydrochloric acid solution (4M) and 1mL of dioxan were slowly added into the

dichloromethane reaction solution at a temperature of 0°C. The reaction solution was stirred

continuously for 30 minutes at a temperature of 0°C, and then stirred continuously for 30

minutes at room temperature. The reactant was subjected to rotary evaporation and

concentration to a full dry state to obtain a white foamy crude product 14 (1.7g, 90%). This

crude product was directly used in the next step without further purification. 'H NMR (CDC 3 ):

6, 8.20 (b, 2H, -NH 2), 5.35 (d, 3H, J=3.OHz, sugar-H-4'), 5.22 (dd, 3H, sugar-H-3'), 4.80 (d,

3H, sugar-H-l'), 4.13 (m, 9H, sugar-2xH-6', sugar-H-2'), 3.94-3.44 (m, 24H, -OCH2, sugar-H

5'), 3.77,3.64,3.46 (m, 6H, -CH2NH-), 2.55,2.43 (m, 6H, -COCH2-),2.15 (s, 9H, -NHCOCH 3 ),

2.09 (s, 2H, -CH2-), 2.05, 1.98, 1.96 (3xs, 27H, -OCOCH3). HRMS (ESI) m/z, C73Hii 9N 70 3 9

(M+H+)/2. Theoretical value: 860.4, measured value: 860.0.

[0084] Compound 21 was prepared according to the following route:

0- 0 OH 0 HO OH DMT-CI HO OH

Pyr / DCM

15 16

0 H 0 H H N1 0 ,R,,- OH + H 2N , N O- T3P, DIPEA N0

O O THF 0

17 18 19

0 H HCI, EtOAc N ,NH rt, 30 min

20

ODMT ODMT

N NH2 HO OH HATU, DIPEA N N OH

0 O DMF 0 0

20 16 21

[0085] Synthesis of compound 16

[0086] 4,4'-dimethoxytrityl chloride (1.8g, 5.3 mmol) was dissolved in 5mL of

dichloromethane, the solution was added dropwise and slowly into an anhydrous pyridine

(1OmL) solution containing 3-hydroxy-2-hydroxymethyl-2-methyl-propanoic acid 15 (0.8g, 5.97 mmol) at room temperature. The solution was stirred continuously for 14 hours at room

temperature. 20mL of water was added to the reaction mixture and extracted with 2x5OmL of

ethyl acetate. The organic phase was subjected to rotary evaporation and concentration to a

semi-dry state, purification was further performed with a silica gel chromatographic column, a

gradient elution was used, initially washed with n-hexane solvent, and then eluted with a mixed

solvent (containing n-hexane/ethyl acetate, 1:1, v/v), the product components were collected,

the solvent was drained under reduced pressure to obtain a yellow solid 16 (1.5g, 58%). The

product 16 was directly used in the next step.

[0087] Synthesis of Compound 19

[0088] Mono-methyl adipate 17 (0.16g, Immol) and N- (tert-butoxycarbonyl)-1,3 diaminopropane N-(3-aminopropyl) tert-butyl carbamate 18 (0.174g, Immol) were dissolved

in 5mL of anhydrous tetrahydrofuran at room temperature. The solution was blended with

0.892mL of 1-propylphosphoric acid cyclic anhydride (0.892mL, 1.5mmol, in 50% (volume

ratio 1:1) ethyl acetate) and 0.522mL of N,N-diisopropylethylamine (DIPEA, 0.522mL,

3mmol). The mixed reaction solution was continuously stirred for 30 minutes at room

temperature, 20mL of ethyl acetate was then added into the reaction solution for dilution, and

20mL of saturated salt solution was added simultaneously, and extracted with 2x2OmL of ethyl

acetate. The organic phase was separated, and dried with anhydrous sodium sulfate, then rotary

evaporated and concentrated to a full dry state, resulting in a yellowish foamed crude product

19 (0.29g, 91%), which was directly used in the next step without further purification. 'H NMR

(CDC 3), 6, 5.89 (b, 1H, -CONH-), 4.97 (b, 1H, -NHCO-), 3.75 (s, 3H, -CH 3), 3.27 (m, 2H),

3.15 (m, 2H), 2.34 (m, 2H), 2.19 (m, 2H), 1.67 (m 2x2H), 1.64 (m, 2H), 1.4 (s, 9H). HRMS

(ESI) m/z, C1 5 H 2 8 N2 0. Theoretical values: 316.39, measured values: 316.40.

[0089] Synthesis of Compound 20

[0090] Compound 19 (0.8g, 2.5 mmol) was dissolved in 5mL of ethyl acetate, the reaction solution was mixed with 3.2mL of aqueous hydrochloric acid solution (4M), and 5mL of

dioxane was further added. The mixed reaction solution was continuously stirred for 30 minutes

at room temperature. After rotary evaporation and concentration, a viscous and thick crude

product 20 (0.5g, 92%) was obtained, which was directly used for the next step.

[0091] Synthesis of compound 21

[0092] The crude product compound 20 (0.252g, immol) was dissolved in 5mL of

dimethylformamide, the reaction solution was blended with 3-0-4,4'-dimethoxytrityl-2

hydroxy-2-methylpropionic acid 16 (0.45g, 0.9 mmol), and added 2-(7-azabenzotriazo)

N,N,N',N'-tetramethylurea hexafluorophosphate (0.46g, 1.2 mmol) and N,N

diisopropylethylamine (0.52 mL) at 0°C and stirred for 20 minutes. The reaction solution was

slowly heated to room temperature and continuously stirred for 14 hours. The reaction solution

was blended with 20mL of saturated sodium chloride solution, and extracted with 2x50mL of

ethyl acetate, the organic phase was dried with anhydrous sodium sulfate and then subjected to

rotary evaporation and concentration in reduced pressure to obtain a crude product 21.

Purification was further performed with a silica gel chromatographic column, a gradient elution

was used, initially washed with ethyl acetate solvent, followed by rinsing with a mixed solvent

(containing ethyl acetate/methanol, 90:10, v/v), the product components were collected, and the solvent was drained under reduced pressure to obtain a compound 21 (0.38g, 61%). 'H NMR

(CDCl3): 6, 8.01 (b, 1H, -CONH-), 7.26 (m, 4H, Trityl), 7.05 (m, 4H, Trityl), 6.67 (m, 4H,

Trityl), 6.48 (b, 1H, -NHCO-), 5.25 (s, 3H, -OCH 3), 3.78 (s, 6H, 2x-OCH3), 3.64 (s, 4H, 2X

CH2-),3.26 (m, 2H, -NHCH2-),3.17 (m, 2H, -CH2NH-), 2.79 (m, 3H, -OCH3 ),2.31 (m, 2H),

2.18 (m, 2H), 1.65-1.56 (m, 6H), 1.25 (s, 3H). HRMS (ESI) m/z, C3 6H 4 6N 2 0, (M+Na').

Theoretical value: 657.34, measured value: 657.40.

[0093] Compound (603A) was prepared according to the following route: ODMT ODMT

O N N OH LiOH,THF/MeOH L N N OH

0 0 0 0 0 C-25°C, 2hrs 21 22

OAc OAc

AcO ON NHAc H OAcOAc 0 0 ODMT 0 0 HATU, DIPEA. DCM AcO O O O- -N 0 H H 14 + 22 NHAc H O N N N OH 0 C-25°C, 2hrs H 0 0 OAc OAc O

O 0 603A AcO O O O N NHAc H

[0094] Synthesis of compound 22

[0095] The purified compound 21 (0.7g, 1.1 mmol) was dissolved in 5mL of anhydrous

methanol, 1.5mL of methanol solution of lithium chloride (2M) was added slowly into the

reaction solution at the temperature of 0°C. The reaction solution was stirred at 0°C for 30 min,

the reaction solution was then heated to room temperature and continuously stirred for 2 hours

at room temperature. After mixing the reaction solution with 2mL of water at room temperature,

the reaction solution was subjected to rotary evaporation and concentration to a semi-dry state

such that the methanol was removed, and separation was performed by using the preparative

reverse phase high pressure liquid chromatography, the mobile phase solvent was methanol and

water (MeOH: H 2 0, 1:1, v/v). The product components were collected, and the solvent was

drained under reduced pressure to obtain a yellow solid compound 22 (0.66g, 93%). 'H NMR

(CDC 3): 6, 7.38 (m, 4H, Trityl), 7.29 (b, 1H, -CONH-), 7.28 (m, 5H, Trityl), 7.16 (b, 1H,

NHCO-), 6.79(m,4H,Trityl),3.76(s,10H,2x-OCH3,2x-CH2),3.71(m,2H,-CH 2-),3.21

(m, 2H, -NHCH2-),2.07 (m, 2H, -CH2NH-), 1.87 (m, 2H), 1.50 (m, 2H), 1.27 (m, 2H), 1.21 (s,

3H). HRMS (ESI) m/z, C 35 H 4 3N 2 0, (M+H++Na'). Theoretical value: 643.72, measured value:

643.20.

[0096] Synthesis of compound (603A)

[0097] The purified compound 22 (0.65g, 1.04 mmol) was dissolved in l5mL of

dichloromethane, and mixed with 0.723mL of N,N-diisopropylethylamine (4.16 mmol) at room

temperature. The mixed solution was blended with the purified compound 14 (1.83g, 1.04

mmol), 2-(7-azabenzotriazo)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.435g, 1.1

mmol) and N,N-diisopropylethylamine (0.723mL, 4.16 mmol) and stirred for 30 minutes under

the temperature of 0°C. The reaction solution was further added with 1mL of N,N

diisopropylethylamine and continuously stirred at 0°C for 1 hour. The reaction temperature was

gradually raised from 0°C to room temperature, then continuously stirred for 2 hours. The

reaction solution was blended with 5mL of saturated sodium chloride solution, and extracted

with 2 X 50mL of dichloromethane, the organic phase was dried with anhydrous sodium sulfate,

followed by rotary evaporation and concentration in reduced pressure to obtain a crude product

603A. Purification was further performed with a silica gel chromatographic column, a gradient

elution was used, initially washed with dichloromethane solvent, then rinsed with a mixed

solvent (containing dichloromethane/methanol/triethylamine, 94:5:1, v/v/v), the product

components were collected, and the solvent was drained under reduced pressure to obtain a

yellow solid compound 603A (1.56g, 65%). 'H NMR (CDCl 3): 6, 7.38 (m, 3H, -NH-), 7.28 (m,

4H, trityl), 7.26 (m, 1H, -NH-), 7.18 (m, 1H, -NH-), 6.84 (m, 5H, trityl), 6.37 (m, 4H, trityl),

5.33 (m, 3H, sugar-H-4'), 5.16 (dd, J=3.4Hz, J=11.3Hz, 3H, sugar-H-3'), 4.77 (d, J=8.4Hz, 3H,

sugar-H-l'), 4.18-4.07 (m, 3x2H, 3xlH, sugar-H-5', sugar-H-6'), 3.94 (m, 3H, sugar-H-2'),

3.77-3.53 (m, 14H), 3.42 (m, 2H, -NHCH2-), 3.30-3.19 (m, 2H, -CH2NH-), 2.42 (m, 2H), 2.19

(m, 4H), 2.15 (s, 9H), 2.07 (m, 2H), 2.05 (s, 9H, 2.01 (s, 9H), 1.96 (s, 9H), 1.20 (s, 3H). HRMS

(ESI) m/z, C 7 H 43N 9 0 4 4 , (M-trityl+H+)/2. Theoretical value: 1,010.55, measured value:

1,010.4.

[0098] Conjugate (603B) triethylamine carboxylate was prepared according to the following

route:

OAc OAc 0 0 AcO O O O N NHAc H OAc OAc 0 0 ODMT AcO O O O N O H H NHAc H N N OH H O O OAc OAc 0 603A AcO NHAc H

DMAP, EtN 3 O O

OAc OAc

AcO ONO O N NHAc H OAc OAc 0 0 ODMT AcO AO O0 -O 0 O 'N N H N ... H 0 0 0Et 3 NH NHAc H O N N N O O~Et3NH H O O O

OAc OAc 0

AcO O 603B NHAc H

[0099] Compound (603A) (1.5g, 0.646mmol) was dissolved in 30 mLof dry dichloromethane, 5 mL of triethylamine was subsequently added. 4-dimethylaminopyridine (0.159g, 1.3 mmol) was dissolved and stirred in the reaction solution, succinic anhydride (0.13g, 1.3 mmol) was also dissolved in the reaction solution with the aid of stirring at room temperature, and the reaction was performed under stirring condition for 8 hours. Succinic anhydride (32mg, 0.32 mmol) was further added and continuously stirred for 14 hours at room temperature. The reacted solution was then poured into a saturated saline solution, extracted with 2x50 mL of dichloromethane, the organic phase was separated, and dried with anhydrous sodium sulfate, then evaporated to a semi-dry state under reduced pressure. Purification was further performed with a silica gel chromatographic column, a gradient elution was used, initially washed with a mixed solvent (containing dichloromethane/methanol/triethylamine, 100:2:1, v/v/v), further rinsed with a mixed solvent (including dichloromethane/methanol/triethylamine, 100:5:1, v/v/v), then rinsed with a mixed solvent (containing dichloromethane/methanol/triethylamine, 100:5:1, v/v/v) to obtain the final product, the solvent was drained under the reduced pressure to obtain a white compound (603B) (1.56g, 65%). 'H NMR (CDCl 3) 6, 'H NMR (CDCl 3): 6, 7.39 (m, 3H, -NH-), 7.28 (m, 5H, trityl), 7.22 (m, 1H, -NH-), 7.10 (m, 1H, -NH-), 6.80 (m, 4H, trityl), 6.37 (m, 4H, trityl), 5.32 (m, 3H, sugar-H-4'), 5.29 (s, 2H), 5.15 (dd, J=3.4Hz, J=1.3Hz,

3H, sugar-H-3'), 4.77 (d, J=8.4Hz, 3H, sugar-H-l'), 4.18-4.07 (m, 3x2H, 3xlH, sugar-H-5',

sugar-H-6'), 3.94 (m, 31, sugar-H-2'), 3.67 (m, 9H), 3.61-3.53 (m, 42H), 3.42 (m, 2H,

NHCH2-),3.30-3.19 (m, 2H, -CH2NH-), 2.42 (m, 2H), 2.19 (m, 4H), 2.15 (s, 911), 2.07 (m, 2H),

2.05 (s, 9H, 2.01 (s, 9H), 1.96 (s, 9H), 1.23 (s, 3H). HRMS (ESI) m/z, C 2 H6 5 N 9 0 4 9 , (M-H+)/2.

Theoretical value: 1,209.27, measured value: 1,209.83.

[00100] Preparation of a conjugate (603C) by connecting the conjugate (603B) to the solid phase carrier according to the following process route: OAc OAc 00 AcO IAcO O'---o O NHAc HN OAc OAc

AcO ON o H H O NHAc H 0 N NN 0 OH H 0 0 0 OAc OAc O

AcO O O N 603B NHAc H

HATU, DIPEA H 2 N-- (CPG, or Nitto-phase, or Aminomethyl polystyrene) ACN

OAc OAc 0 0 AcO O,,N,-- O N NHAc H OAc OAc O 0 0 ODMT AcO N 0 H HO NHAc H N N N 0NH H 0 0 0 OAc OAc 0 O 0 603C AcO ~ O ,--,'N NHAc H

[00101] The conjugate (603B) (50mg, 0.021mmol) and 2-(7-azabenzotriazo)-N,N,N',N'

tetramethylurea hexafluorophosphate (10mg, 0.026 mmol) were dissolved in 1.25 mL of

anhydrous acetonitrile at room temperature. N,N-diisopropylethylamine (10pL) was added into

the reaction solution, after all reagents were dissolved, 125 mg of long-chain amino alkane glass

sand (500°A, native lcaa-CPG, Chemgenes, USA) was added into the reaction solution. The

solid-phase and liquid-phase were rotated and stirred for 300 rpm at room temperature. After

the reaction lasted for 2 hours, the residue liquid was filtered, the long-chain amino alkane glass sand (solid phase carrier) was washed with acetonitrile for three times (3x1mL). 0.5mL of a tetrahydrofuran solution of capping reagent A (acetic anhydride) having a concentration of 10% (v/v) and 0.5mL of capping reagent B (a mixed solution of N-methylimidazole, pyridine and acetonitrile at a concentration of 15:10:75, v/v/v) and the long-chain amino alkane glass sand were rotated and stirred for 1 hour at room temperature. The reaction solution was filtered, the long-chain amino alkane glass sand (solid phase carrier) was rinsed with acetonitrile for three times, then dried under reduced pressure for 2 hours by using an oil vacuum pump, a glass sand solid phase carrier (603C, 130mg) was obtained. The solid phase carrier 603C (8.3mg) was weighed and added into 1OOmL of a dichloromethane solution of trichloroacetic acid having a concentration of 3%, then rotated and stirred for 30 seconds and stood still for 1 minute. The supernatant was taken to measure the visible light absorption at 498nm, its light absorbance was 0.309, a loading capacity of the conjugate 603C (i.e., a loading capacity of (603B) on CPG) was calculated as 53.25 [mol/g.

[00102] Preparation of sense and antisense strands of siRNA (preparation of siRNA drugs)

[00103] Pursuant to the solid phase synthesis method of phosphoramidites, the nucleoside monomers were linked one by one according to the sequence along the direction from 3'-5' according to the above sequence. Each linking with a single nucleoside monomer included the four-step reaction of deprotection, coupling, capping and oxidation.

[00104] Formulation of solid phase synthesis reagent:

[00105] The deprotection reagent was a dichloromethane solution of trichloroacetic acid (TCA) or dichloroacetic acid (DCA) having a concentration of 3% (v/v). The nucleoside monomers were dissolvedin anhydrous acetonitrile having a concentrationof 0.05-0.1M, whichwas added with a small amount of molecular sieve 3A° for anhydrous treatment. The coupling activator was anhydrous acetonitrile of 5-ethylthio-1H-tetrazole having a concentration of 0.25M or 0.45M, alternatively the activator may be selected from1H-hetrazole, 5-benzyylthio-1H tetrazole and 4,5-dicyanoimidazole. The capping reagent A was a tetrahydrofuran solution of acetic anhydride having a concentration of 10% (v/v). The capping reagent B was a mixed solvent of N-methylimidazole, pyridine and acetonitrile having a concentration of 15:10:75 (v/v/v). The oxidizing reagent was the water and pyridine solution of iodine having a concentration of 0.05M (95wt% of an aqueous pyridine solution). The sulfurization reagent was

N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3- thione having a concentration

of 0.05M (pyridine/acetonitrile, 2:3, v/v). The deprotection reagent was concentrated aqueous

ammonia with a concentration of 28 wt.%.

[00106] Wherein the solid phase carrier for solid phase synthesis of nucleotides was commercially available and commonly used solid phase carrier (NittoPhase@ HL UnyLinker TM

300; or 500°A, native lcaa-CPG, Chemgenes, USA).

[00107] Steps of solid phase synthesis:

[00108] A solid phase carrier was blended with a dichloromethane solution of trichloroacetic acid having a concentration of 3% (v/v) with a molar ratio of 1:30 on a synthesizer. The reaction

was performed for 1.5 minutes at room temperature, and the operation was repeated for three

times, the dropwise adding of a deprotection reagent was stopped when the color of elution

solution of solid phase carrier was changed from red color to colorless. After repeated washing

with anhydrous acetonitrile, the nucleoside monomer and activator (coupling activator, 5

ethylthio tetrazole) were added (at a molar ratio of nucleoside monomer to activator of 1:20),

and a molar ratio of the solid phase carrier and nucleoside monomer were 1: (5-6). The reagents

were at room temperature and the time of the solid phase reaction was 3-4 minutes for one cycle,

and after two cycles, the reaction was stopped. After washing with anhydrous acetonitrile, the

oxidizing reagent solution was added, a molar ratio of the solid phase carrier and the oxidizing

reagent were 1:6. The reaction time of the oxidizing reagent and the solid phase carrier at room

temperature was about 2 minutes, the operation was repeated twice. After the coupling reaction,

if a sulfidation reaction step was required, the sulfidation reagent solution was added, a molar

ratio of the solid phase carrier and the sulfidation reagent was 1:6. The reaction time of the

sulfidation reagent and the solid phase carrier at room temperature was about 4-5 minutes, and

the operation was repeated twice. The capping protection reaction was performed by adding the

capping reagent, a molar ratio of the solid phase carrier and the capping reagent was 1:80. The

reaction time of the capping reagent and the solid phase carrier at room temperature was about

1-2 minutes, and the operation was repeated twice. The aforementioned deprotection, coupling,

oxidizing and capping steps were cycled until the coupling of the last nucleotide was completed.

The solid phase carrier loaded with the sense strand or the antisense strand of the nucleic acid

sequence was transferred into a vial bottle, an aqueous ammonia solution having a concentration of 28% was added, and the glass cap was screwed tightly for sealing, the base protecting group of the sense strand or the antisense strand was hydrolyzed and removed at the temperature of

55°C, while the sense strand or the antisense strand was hydrolyzed and separated from the

solid phase carrier. The reaction was run for 16 hours. The resulting solution of the small nucleic

acid strand was filtered and separated from the solid phase carrier. After concentration, a crude

product of the small nucleic acid strand was obtained.

[001091 Purification, separation and desalting by using the preparative high pressure liquid chromatography

[00110] Small nucleic acid was purified by gradient elution of NaBr using a preparative type anion exchange chromatographic column (Source 15Q). Mobile phase A: 20mM sodium

phosphate (pH 8.0), mobile phase B: 20mM sodium phosphate (pH 8.0), IM sodium bromide

in aqueous acetonitrile having a concentration of 10%. The column temperature was 65°C. The

flow rate was 1OmL/min. The elution gradient was initiated a mobile phase A, followed by a

change in mobile phase B from 0% to 20% at 12 minutes. The mobile phase B increased from

20% to 50% in the subsequent 15 minutes. The eluent was collected, and subjected to

component analysis and component combination. Desalting was performed by using a reverse

phase chromatography purification column, or a dialysis desalting process. The eluent was

subjected to concentration and freeze drying to obtain purified small nucleotides. For the

synthesized sense and antisense strands, purity was detected using the anion exchange liquid

chromatogram (AEX-HPLC), and the full sequence molecular weight was identified and

analyzed by reverse phase liquid chromatography-mass spectrometry (LC-MS), the measured

value for molecular weight was consistent with the theoretical value to confirm success in

synthesizing the nucleic acid sequence.

[00111] Annealing

[00112] The synthesized sense strand (S strand) and antisense strand (AS strand) were blended

at an equimolar ratio in a normal saline for injection, heated at a temperature of 90°C for 5

minutes, then cooled slowly to room temperature and stored in a refrigerator at 4°C for 12 hours

to form double-stranded structure through hydrogen bond, the siRNA drugs 30-34 were

obtained (the sequences and modifications of nucleotides were shown in Table 3).

[00113] Table 3

No. Sense strand sequence (5'-3') Antisense strand sequence (5'-3')

30 GsCsUCGGUfGAfGUfGAUGGCAGAA-TriGalNAc (17) UsUfsCUfGCfCAUCACUCfACfCGAGCsUsU (18+UU) 31 GsGsUCACCfGAfCUtJCGAGAAUGU-TriGaINAc (25) AsCfsAUfJCtJCGAAGUCfGGtGACCsUsU (26+UU) 32 CsCsCAAGCfAAfGCfAGACAUUU-TriGaINAc (31) AsAfsAUfGUfCUGCUUfGCt1JUGGGsUsU (32+UU)

33 GsCsAGGAAfCUfGAfGCCAGAAA-TriGaINAc (37) UsUfsUCfUGfGCUCAGfUUfCCUGCsUsU (38+UU) 34 GsCsCAAGCfCUfCUtJCUUACUUCA-TriGaINAc (45) UsGfsAAfGUfAAGAAGAGfGCfUUGGCsUsU (46+UU)

[00114] In the sequences shown in Table 3, the lowercase letter f indicates that the nucleotide adjacent to the left side of the letter f is a 2'-fluoro modified nucleotide (i.e., the 2'-OH of the

pentose in the nucleotide is substituted by fluorine); the lowercase letter s indicates that two

nucleotides adjacent to the left side and right side of the letter s are connected through a

thiophosphate diester bond (i.e., the non-bridging oxygen atom in the phosphodiester bond is

substituted by sulfur atom); the underlined nucleotide indicates that the 2' hydroxyl group of

the nucleotide is substituted by methoxyl group. TriGaNAc denotes a target group and a linkage

group for target delivery (i.e., a moiety of formula (603) other than Nu).

[00115] The sequence and structure of the synthesized siRNA drugs were confirmed by LC MS analysis data. The modified siRNA in Table 3 was provided with a more stable nucleic acid

structure as well as high specificity and high affinity of base pairing. In addition, the nucleic

acid having the above modification exhibited more excellent in vivo inhibitory effect, and can

further reduce the in vivo immunogenicity of the nucleic acid of the present disclosure.

[00116] (II) The male humanized PCSK9 mice (4-6 weeks old, Shanghai Model Organisms Technology Co., Ltd.) were randomly grouped by weight, 3 mg/kg of siRNA drug (the dosage

was based on siRNA alone) was injected into the mice on the zero day, the 2nd day and the 4th

day, serum was taken on the zero day, the 7th day, the 1 4 th day, the 21" day and the 2 9 th day, the

PCSK9 concentration in serum was measured (control group was injected with normal saline).

The serum human PCSK9 concentration measurement was accomplished by using a human

PCSK9 ELISA kit (R&D Systems, Minneapolis, MN, USA, cat#DPC900), and the

experimental procedures were performed as recommended by the manufacturer. FIG. 1 showed

the effect that siRNA reduced the serum PCSK9 protein in humanized PCSK9 mice.

[00117] Meanwhile, the mice were killed at the 2 9 th day and the liver tissue was removed, RNA

was extracted according to the operation instruction of RNAeasy Mini kit (QIAGEN, Article

No. 74104). RT-PCR was performed according to the recommendation of High Capacity cDNA

Reverse Transcription Kits (Thermo Fisher, Article No. 4368814), 1 g of RNA was contained

internal reference gene (mouse HPRT1) was Mm03024075_ml (Thermo Fisher Scientific,

2A-AACt, and the mouse HPRT1 gene expression was used as the internal reference. The

expression level of PCSK9 gene in liver was expressed as a percentage of treated group vs.

control group (Table 2).

[00118] The male humanized PCSK9 mice (4-6 weeks old, Shanghai Model Organisms Technology Co., Ltd.) were randomly grouped by weight, the siRNA drug 34 was injected into

the mice on the zero day, serum was taken on the 14th day, the PCSK9 concentration in serum

was measured (the control group was the PCSK9 concentration of serum in mice injected with

normal saline). The serum human PCSK9 concentration measurement was accomplished by

using a human PCSK9 ELISA kit (R&D Systems, Minneapolis, MN, USA, cat#DPC900), and

the experimental procedures were performed as recommended by the manufacturer. FIG. 3

showed that siRNA dose-dependently decreased the serum PCSK9 concentration of humanized

PCSK9 mice.

[00119] All publications, patents and patent applications mentioned in the specification are

incorporated herein by reference, the extent is same as that each individual publication, patent

and patent application is specifically and individually incorporated herein by reference.

[00120] The above content describes in detail the preferred embodiments of the present

disclosure, but the present disclosure is not limited thereto. A variety of simple modifications

can be made in regard to the technical solutions of the present disclosure within the scope of

the technical concept of the present disclosure, including a combination of individual technical

features in any other suitable manner, such simple modifications and combinations thereof shall

also be regarded as the content disclosed by the present disclosure, each of them falls into the

protection scope of the present disclosure.

Claims

Claims 1. A nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand

contains a sequence having at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:3,

SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,

SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID

NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ

ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,

SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55 or SEQ ID NO:57; the antisense strand

contains a sequence having at least 80% sequence identity to SEQ ID NO:2, SEQ ID NO:4,

SEQIDNO:6,SEQIDNO:8,SEQIDNO:10,SEQIDNO:12,SEQIDNO:14,SEQIDNO:16,

SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID

NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,

SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO: 58.
2. The nucleic acid of claim 1, wherein at least 80% sequence identity refers to that there is one

different base between the sequences; the different base in the sense strand is the last base of

the sense strand, the different base in the antisense strand is the first base of the antisense strand.
3. The nucleic acid of claim 1, wherein the sense strand has 16-30 nucleotides; and/or the

antisense strand has 16-30 nucleotides.
4. The nucleic acid of claim 1, wherein the nucleic acid is at least one selected from siRNA-1

having a sense strand sequence of SEQ ID NO:1 and an antisense strand sequence of SEQ ID

NO:2, siRNA-2 having a sense strand sequence of SEQ ID NO:3 and an antisense strand

sequence of SEQ ID NO:4, siRNA-3 having a sense strand sequence of SEQ ID NO:5 and an

antisense strand sequence of SEQ ID NO:6, siRNA-4 having a sense strand sequence of SEQ

ID NO:7 and an antisense strand sequence of SEQ ID NO:8, siRNA-5 having a sense strand

sequence of SEQ ID NO:9 and an antisense strand sequence of SEQ ID NO:10, siRNA-6 having a sense strand sequence of SEQ ID NO:11 and an antisense strand sequence of SEQ ID NO:12, siRNA-7 having a sense strand sequence of SEQ ID NO:13 and an antisense strand sequence of SEQ ID NO:14, siRNA-8 having a sense strand sequence of SEQ ID NO:15 and an antisense strand sequence of SEQ ID NO:16, siRNA-9 having a sense strand sequence of SEQ ID NO:17 and an antisense strand sequence of SEQ ID NO:18, siRNA-10 having a sense strand sequence of SEQ ID NO:19 and an antisense strand sequence of SEQ ID NO:20, siRNA-11 having a sense strand sequence of SEQ ID NO:21 and an antisense strand sequence of SEQ ID NO:22, siRNA-12 having a sense strand sequence of SEQ ID NO:23 and an antisense strand sequence of SEQ ID NO:24, siRNA-13 having a sense strand sequence of SEQ ID NO:25 and an antisense strand sequence of SEQ ID NO:26, siRNA-14 having a sense strand sequence of SEQ ID NO:27 and an antisense strand sequence of SEQ ID NO:28, siRNA-15 having a sense strand sequence of SEQ ID NO:29 and an antisense strand sequence of SEQ ID NO:30, siRNA-16 having a sense strand sequence of SEQ ID NO:31 and an antisense strand sequence of SEQ ID NO:32, siRNA-17 having a sense strand sequence of SEQ ID NO:33 and an antisense strand sequence of SEQ ID NO:34, siRNA-18 having a sense strand sequence of SEQ ID NO:35 and an antisense strand sequence of SEQ ID NO:36, siRNA-19 having a sense strand sequence of SEQ ID NO:37 and an antisense strand sequence of SEQ ID NO:38, siRNA-20 having a sense strand sequence of SEQ ID NO:39 and an antisense strand sequence of SEQ ID NO:40, siRNA 21 having a sense strand sequence of SEQ ID NO:41 and an antisense strand sequence of SEQ ID NO:42, siRNA-22 having a sense strand sequence of SEQ ID NO:43 and an antisense strand sequence of SEQ ID NO:44, siRNA-23 having a sense strand sequence of SEQ ID NO:45 and an antisense strand sequence of SEQ ID NO:46, siRNA-24 having a sense strand sequence of SEQ ID NO:47 and an antisense strand sequence of SEQ ID NO:48, siRNA-25 having a sense strand sequence of SEQ ID NO:49 and an antisense strand sequence of SEQ ID NO:50, siRNA 26 having a sense strand sequence of SEQ ID NO:51 and an antisense strand sequence of SEQ ID NO:52, siRNA-27 having a sense strand sequence of SEQ ID NO:53 and an antisense strand sequence of SEQ ID NO:54, siRNA-28 having a sense strand sequence of SEQ ID NO:55 and an antisense strand sequence of SEQ ID NO:56, siRNA-29 having a sense strand sequence of SEQ ID NO:57 and an antisense strand sequence of SEQ ID NO: 58.
5. The nucleic acid of claim 1, wherein the nucleic acid is selected from a siRNA-23 having a

sense strand sequence of SEQ ID NO: 45 and an antisense strand sequence of SEQ ID NO: 46,

and having the modifications at the nucleotide as follows:

5'-GsCsCAAGCfCUfCUfUCUUACUUCA -3'

5'-UsGfsAAfGUfAAGAAGAGfGCfUUGGCsUsU-3'

wherein the lowercase letter f indicates that the nucleotide adjacent to the left side of the letter

f is a 2'-fluoro modified nucleotide; the lowercase letter s indicates that two nucleotides adjacent

to the left side and right side of the letter s are connected through a thiophosphate diester bond;

the underlined nucleotide indicates that the 2' hydroxyl group of the nucleotide is substituted

by methoxyl group.
6. A targeted drug delivery system comprising a target group, a linkage group, and the nucleic

acid of claim 1 connected with the target group via the linkage group.
7. The targeted drug delivery system of claim 6, wherein the targeted drug delivery system has

a structure as shown below: OH OH

HO VO O O N O NHAc H OH OH 0 Ou H c O O N O N H NHAc H H 0 O

OH OH 0

HON HO O -O" O - N NHAc H

Formula (603).
8. A pharmaceutical composition comprising the nucleic acid of claim 1, or the targeted drug

delivery system of claim 6, and a pharmaceutically acceptable carrier.
9. A method of inhibiting PCSK9 comprising administering the nucleic acid of claim 1 to a

patient suffering from the hyperlipidemia related diseases.
10. The method of claim 9, wherein the hyperlipidemia related diseases is at least one selected

from coronary atherosclerosis heart disease, ischemic stroke, arteriosclerosis cerebral infarction,

hypertension, diabetes mellitus, nephropathy and peripheral vascular diseases.
11. The method of claim 9, wherein the patient is a human patient.