siRNA for targeted regulation of PCSK9 gene expression and its application

Abstract

The present disclosure relates to siRNA and its use for targeting control of PCSK9 gene expression.Experimental results show that oligonucleotide sequences with some alternating modifications and specific template modifications can significantly inhibit PCSK9 expression, and can be used to develop drugs for the treatment of PCSK9-related diseases.

Description

[Technical Field]

[0001] This application claims priority from a Chinese patent application, application number 202311463375.7, filed with the State Intellectual Property Office of the People's Republic of China on November 6, 2023, with the invention title "siRNA for targeted control of PCSK9 gene expression and its application," the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present disclosure relates to the technical field of nucleic acid modification, and in particular to small interfering ribonucleic acids (siRNAs) modified by various chemical methods and the use of siRNAs in the manufacture of medicaments for the treatment of diseases associated with PCSK9 gene expression. [Background technology]

[0003] Nucleic acid drugs, especially oligonucleotide drugs, have been widely applied due to their easy synthesis and relatively high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and nucleic acid aptamers.

[0004] Oligonucleotides are short DNA or RNA molecules or oligomers that can easily bind to their complementary oligonucleotides, DNA or RNA, in a sequence-specific manner to form double-stranded or rarely observed higher-order hybrids. Due to this fundamental property, oligonucleotides have a wide range of applications in genetic testing, research and medicine. In nature, oligonucleotides are usually small RNA molecules that play a role in regulating gene expression, or they are intermediates obtained from the degradation of larger nucleic acid molecules.

[0005] RNA interference (RNAi) is a natural defense mechanism against foreign genes. siRNAs can knock out target genes by recognizing specific sequences and degrading the target mRNA.

[0006] Classical RNAi molecules are composed of a typical 19+2 nucleotide polymer structure (a double helix consisting of a 21-nucleotide RNA molecule and 19 nucleotide molecules corresponding to the nucleic acid bases, including a two-nucleotide 3' overhang). One strand of the siRNA (the guide strand or antisense strand) is complementary to the target gene transcript mRNA, while the other strand is labeled as the passenger strand (or sense strand). The antisense siRNA guides the Argonaute protein (AGO2) to complement the target transcript and becomes part of the RNA-induced silencing complex (RISC). The antisense siRNA is perfectly complementary to the target, resulting in cleavage of the target transcript at positions 10-11 opposite the guide strand (antisense strand) under the catalysis of the AGO2 protein.

[0007] siRNAs perform their function by completing Watson-Crick base pairing with mRNA, whereas small molecules and monoclonal antibodies must recognize the complex spatial and 3D structure of specific proteins. This gives siRNAs an inherent advantage over small molecules and monoclonal antibodies. Many diseases cannot be treated with small molecules or monoclonal antibodies because the target molecules lack the molecular structure required for high activity and affinity and binding specificity. The mechanism of action of siRNA drugs allows them to control the expression of target proteins at the genetic level, providing greater target specificity than small molecules or antibody drugs. Based on the principle of complementary base pairing, siRNAs offer a broader therapeutic range, are easier to design, and require shorter research and development periods.

[0008] Oligonucleotides can bind to complementary RNA strands in a sequence-specific manner and induce RNase H cleavage of target RNA after hybridization. In natural oligonucleotides, nucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly susceptible to nucleases. Natural, unmodified, and unmodified oligonucleotide drugs are prone to rapid degradation by nucleases in vivo, resulting in low activity and poor drug potential. Chemical modifications to the oligonucleotide structure can effectively enhance their activity, improving their stability against nucleases and their affinity for RNA, thereby better promoting endocytosis and tissue targeting and effectively controlling target gene expression.

[0009] Depending on the basic structure of the oligonucleotide (base, sugar ring, phosphate backbone, and terminus), chemical modification can be carried out in four parts. 1) Base modifications: These are mainly divided into three types: purine modifications, pyrimidine modifications, and base substitutions. Purine modifications include N6-methyladenosine, N1-methyladenosine, and 7-methylguanylate. Pyrimidine modifications include 3-methyluridine, 5-methyluridine, 5-methylcytidine, N4-acetylcytidine, pseudouridine, thiouridine, propyneuridine, and dihydrouridine. 2) Sugar ring modifications: These are mainly divided into sugar ring modifications and substitutions. Sugar ring modifications include 2'-modifications, 4'-modifications, 5'-modifications, isomerization modifications, and combinations thereof. The most common 2'-modifications in siRNA are 2'-OMe (2'-methoxy) and 2'-F (2'-fluoro). siRNAs modified with both 2'-OMe and 2'-F have higher Tm values, stronger serum stability, and better activity than native siRNAs. 3) Modifications of the phosphate backbone: The main modifications include thiophosphate, methylphosphate, selenophosphate, methylborylphosphate, dithiophosphate, and bridging oxygen atom substitution in the phosphodiester linkage region with a sulfur atom, as well as substitution of the entire internucleoside phosphate group with a group that does not contain a phosphorus atom (e.g., substitution of a P atom with a C, S, or N atom to form a guanidine group, S-methylthiourea, etc.). 4) Terminal modifications: Covalent conjugation of special groups to the 5' and / or 3' termini of the sense strand, and phosphorylation modifications of the 5' end of the antisense strand. All commercially available oligonucleotide drugs are chemically modified. Since the first nucleic acid drug, Fomivirsen (Vitravene), was approved for listing in 1998, the chemical modification technology for nucleic acid drugs has been continuously improved. To date, 18 types of nucleic acid drugs are commercially available worldwide.

[0010] [Table 1] TIFF2025541705000002.tif49168

[0011] The primary treatment strategy for hypercholesterolemia is to lower low-density lipoprotein cholesterol (LDL-C), which contributes to atherosclerosis, and to raise high-density lipoprotein cholesterol (HDL-C), which has potential cardioprotective effects. Clinical studies involving approximately 170,000 patients have shown that a 1.0 mmol / L reduction in LDL-C levels corresponds to a 20% reduction in the average annual incidence of major cardiovascular events. The 2019 edition of the "Guidelines for the Basic Management of Dyslipidemia" recommends LDL-C-lowering medications, primarily including statins, cholesterol absorption inhibitors, Robutecol, and PCSK9 inhibitors, with statins being the first-line treatment. The 2018 edition of the "Consensus of Chinese Experts on the Screening and Treatment of Familial Hypercholesterolemia" recommends the combination of statins with ezetimibe and PCSK9 inhibitors.

[0012] PCSK9 is a serine protease encoded by the PCSK9 gene and is primarily produced in the liver. PCSK9 binds to LDL receptors (LDL-R) on the surface of hepatocytes and degrades them. LDL-R binds to LDL-C in plasma and transports it to the liver (where it is processed and excreted in bile). Therefore, degradation of LDL-R leads to an increase in plasma LDL-C levels. PCSK9 inhibitors reduce the expression level of PCSK9 and increase LDL-R levels on the surface of hepatocytes, thereby reducing plasma LDL-C levels and achieving the goal of lipid lowering.

[0013] There is a need in the art to develop further agents to regulate PCSK9 gene expression in order to effectively treat hypercholesterolemia and dyslipidemia. Summary of the Invention [Problem to be solved by the invention]

[0014] In this disclosure, a series of unique siRNA sequences are designed against the PCSK9 mRNA sequence with alternating and specific template modifications.

[0015] Generally, siRNA is modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) in the monomer. However, even if only the combination of the above two monomer modifications is considered, for an siRNA having a total of 44 bases in the sense and antisense strands, 2 44 Furthermore, when the placement of different terminal thiol modifications is also taken into account, the number of possible modification schemes becomes enormous.

[0016] When different modification forms are used for the same siRNA sequence, the activity is very different, and when the same modification form is used for different siRNAs, the activity is also very different.In addition, although there are some modification principles for siRNA modification design, it has been reported that the activity cannot be accurately predicted from the modification form, that is, there is no definite relationship between the modification form and activity.Therefore, it is very difficult to select the modification scheme with high activity from the combination of countless possible modifications.

[0017] In the present disclosure, several alternatively modified sequences and specially modified sequences that have a significant inhibitory effect on PCSK9 gene expression are selected by chemically modifying designed siRNA sequences.

[0018] In one aspect, the present disclosure provides a double-stranded RNAi agent for reducing PCSK9 expression, comprising any one selected from the following double-stranded oligonucleotides in which the sense strand and the antisense strand match: (1) a double-stranded oligonucleotide, the sense strand of which has the sequence set forth in SEQ ID NO. 1 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence set forth in SEQ ID NO. 13 or a fragment thereof, or a modified version of said sequence or said fragment; (2) a double-stranded oligonucleotide, the sense strand of which has the sequence set forth in SEQ ID NO. 2 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence set forth in SEQ ID NO. 14 or a fragment thereof, or a modified version of said sequence or said fragment; (3) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 3 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 15 or a fragment thereof, or a modified version of said sequence or said fragment; (4) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 4 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 16 or a fragment thereof, or a modified version of said sequence or said fragment; (5) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified version of said sequence or said fragment; (6) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 6 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 18 or a fragment thereof, or a modified version of said sequence or said fragment; (7) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 7 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 19 or a fragment thereof, or a modified version of said sequence or said fragment; (8) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified version of said sequence or said fragment; (9) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 9 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 21 or a fragment thereof, or a modified version of said sequence or said fragment; (10) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 10 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 22 or a fragment thereof, or a modified version of said sequence or said fragment; (11) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 11 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 23 or a fragment thereof, or a modified version of said sequence or said fragment; (12) A double-stranded oligonucleotide, wherein the sense strand has the sequence set forth in SEQ ID NO. 12 or a fragment thereof, or a modified sequence of said sequence or said fragment, and the antisense strand has the sequence set forth in SEQ ID NO. 24 or a fragment thereof, or a modified sequence of said sequence or said fragment.

[0019] In another embodiment, the present disclosure also provides a conjugate for reducing PCSK9 expression, comprising the double-stranded RNAi agent and a ligand conjugated thereto.

[0020] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the double-stranded RNAi agent or conjugate and a pharmaceutically acceptable carrier.

[0021] In another aspect, the disclosure also provides the use of the double-stranded RNAi agent, conjugate, or composition in the manufacture of a medicament for the treatment of a PCSK9-related disease.

[0022] In another aspect, the present disclosure also provides methods of treating or preventing a disease or condition that can be modulated by downregulating PCSK9 gene expression.

[0023] The beneficial effects achieved by the present disclosure include at least the following: (1) The unmodified base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83 have significant inhibitory effects on PCSK9, with inhibition rates exceeding 60%. (2) Using multiple modified sequences, the inhibition rate can reach up to 90% or more. Furthermore, when the modified sequences are conjugated with GalNAc compounds, they can be efficiently delivered to the animal liver, where they significantly inhibit PCSK9 gene expression, significantly reduce serum low-density lipoprotein cholesterol (LDL-C) levels, and significantly reduce serum total cholesterol (TC) levels. (3) Each sequence modified with the alternating modifications and modification templates of the present disclosure exhibits significantly improved inhibitory activity against PCSK9, up to a 40% increase, compared to unmodified sequences with identical or only minor differences from sequences disclosed in the prior art. (4) This disclosure demonstrates that siRNAs with similar sequences, whether unmodified or alternatively modified, exhibit significantly different activities. For example, the unmodified base sequence 5 exhibited a 51.2% higher inhibitory rate than base sequence 4, while the alternatively modified sequence P92-si5 exhibited a 61.8% higher inhibitory rate than P92-si4, demonstrating significant improvements. (5) Furthermore, the present disclosure has found that alternating modifications of different sequences have varying effects on activity, such as a significant improvement in inhibition rate (12.4% improvement in inhibition rate) for sequences obtained by alternating modification of base sequence 5 compared to the unmodified sequence, and a minimal change in inhibition rate (negligible change in inhibition rate) for sequences obtained by alternating modification of base sequences 4, 22, and 52 compared to the unmodified sequence. [Brief explanation of the drawings]

[0024] In order to more clearly describe the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments are briefly introduced below. It is obvious that the drawings in the following description only relate to some embodiments of the present disclosure and do not limit the present disclosure. [Figure 1] The present invention shows an alternately modified sequence that has a significant inhibitory effect on the PCSK9 gene, with an inhibition rate of over 50%. [Figure 2] The alternating modified sequences exhibit an inhibitory rate of 30% to 50% against the PCSK9 gene. [Figure 3] 1 shows alternatively modified sequences that have an inhibition rate of less than 30% against the PCSK9 gene. [Figure 4] 1 shows alternatively modified sequences that have an inhibition rate of less than 30% against the PCSK9 gene. [Figure 5] The unmodified sequence has a significant inhibitory effect on the PCSK9 gene, with an inhibition rate of over 50%. [Figure 6] Unmodified sequences with an inhibition rate of less than 40% against the PCSK9 gene are shown. [Figure 7] 1 shows the inhibition rates of base sequence 5 modified with different templates against the PCSK9 gene. [Figure 8] The inhibition rate of the PCSK9 gene of sequences with template modifications, anti-off-target and / or 5'-E-VP modifications in base sequences 5, 51, 81 and 84 is shown. [Figure 9]1 shows the inhibition rate of PCSK9 protein in animal serum using different modified forms of base sequences 5, 51, 81, 82 and 84 and after conjugation with GalNAc compounds. [Figure 10] 1 shows the reduction level of low density lipoprotein cholesterol (LDL-C) in animal serum using different modified forms of base sequences 5, 51, 81, 82 and 84 and after conjugation with GalNAc compounds. [Figure 11] The reduction level of total cholesterol (TC) in animal serum is shown using different modified forms of base sequences 5, 51, 81, 82 and 84 and after conjugation with GalNAc compounds. DETAILED DESCRIPTION OF THE INVENTION

[0025] To facilitate understanding of this disclosure, several terms are first defined. It should be noted that when a value or range of values ​​for a parameter is listed, it is intended to indicate that intermediate values ​​and ranges of these recited values ​​are also part of this disclosure.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or to more than one element, such as a plurality of elements.

[0027] The term "including" means, and is used alternatively with, the phrase "including but not limited to."

[0028] The term "or" means, and is used alternatively with, the term "and / or," unless the context clearly indicates otherwise.

[0029] As used herein, the term "about" or "approximate" as applied to one or more target values ​​refers to a value similar to the reference value. In some embodiments, unless otherwise specified or further apparent from the context, the term "approximate" or "about" refers to a range of values ​​that falls within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the reference value (as long as it does not exceed 100% of the possible values).

[0030] As used herein, "PCSK9" refers to the preproprotein convertase subtilisin Kexin 9 gene or protein.

[0031] "G," "C," "A," and "U" generally refer to nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" can be used interchangeably herein and refer to deoxyribonucleotides whose nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides (as described in more detail below) or alternative replacement moieties. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted for other moieties without substantially altering the base-pairing properties of an oligonucleotide (including nucleotides having such replacement moieties). For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted in the nucleotide sequences of the present disclosure with nucleotides containing, for example, inosine, and sequences containing such substitutions are described in the examples of the present disclosure.

[0032] The terms "RNAi agent," "iRNA," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to terms including RNA agents, as defined herein, that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNAi directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). RNAi controls (e.g., suppresses) the expression of PCSK9 in cells, such as cells within a subject (e.g., a mammalian subject). RNAi molecules include single-stranded RNAi molecules and double-stranded siRNAs, as well as short hairpin RNAs (shRNAs).

[0033] The term "small interfering ribonucleic acid" or "siRNA" refers to a small interfering ribonucleic acid (RNAi) molecule. It belongs to a type of double-stranded RNA molecule and is also referred to in the art as short interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleosides in length. The antisense strand is complementary to a target nucleic acid (preferably a mature mRNA sequence) (e.g., with at least 95% complementarity, e.g., complete complementarity), and the sense and antisense strands are complementary to form a duplex or double-stranded region. The siRNA strands may form a blunt-ended duplex, or preferably, may form 3' overhangs, such as 1, 2, or 3 nucleosides, at the 3' ends of the sense and antisense strands, which can form RISC substrates in vivo, similar to the products produced by Dicer. Effective extension forms of Dicer substrates are described in U.S. Patent Nos. 8,349,809 and 8,513,207, which are incorporated herein by reference. In some embodiments, both the sense strand and the antisense strand have a 2-nt 3' overhang. Thus, the double-stranded region may have a length of, for example, 17 to 25 (e.g., 21 to 23) nucleotides.

[0034] The term "antisense strand" refers to the strand of RNAi (e.g., dsRNA) that comprises a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) defined herein. If the complementary region is not completely complementary to the target sequence, mismatches may be present within the internal or terminal regions of the molecule. Typically, mismatches are most tolerated within terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0035] As used herein, the term "sense strand" refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0036] As used herein, the term "inhibition / suppression" can be used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any degree of inhibition.

[0037] As used herein, the term "inhibition of PCSK9 expression" refers to inhibiting the expression of any PCSK9 gene (e.g., mouse PCSK9 gene, rat PCSK9 gene, monkey PCSK9 gene, human PCSK9 gene, etc.), and variants (e.g., naturally occurring variants, etc.) or mutants of the PCSK9 gene. Thus, the PCSK9 gene may be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the case of a genetically engineered cell, cell population, or organism.

[0038] "Inhibition of PCSK9 gene expression" includes any degree of inhibition of the PCSK9 gene, including, for example, at least partial inhibition of PCSK9 gene expression, e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0039] PCSK9 gene expression can be assessed based on the level of any variable associated with PCSK9 gene expression, such as PCSK9 mRNA level, PCSK9 protein level, or serum lipid level. Inhibition can be assessed by a reduction in one or more of these variables, either absolute or relative to a control level. The control level can be any type of control level utilized in the art, such as a baseline level before dosing, or a level measured from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer control or an inactive agent control only).

[0040] As used herein, a "patient" or "subject" refers to a human or non-human animal, preferably a mammal such as a monkey, and most preferably a human.

[0041] As used herein, "PCSK9-related disease" refers to any disease associated with the PCSK9 gene or protein. Such diseases can be caused, for example, by overproduction of PCSK9 protein, PCSK9 gene mutation, abnormal cleavage of PCSK9 protein, or abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipidemias such as hyperlipidemia, and other forms of dyslipidemia such as hypercholesterolemia and hypertriglyceridemia, as well as pathological conditions associated with these abnormalities, such as heart and circulatory system diseases.

[0042] As used herein, a "therapeutically effective amount" refers to the amount of an RNAi agent that, when administered to a patient for the treatment of a PCSK9-related disease, is sufficient to effect treatment of the disease (e.g., attenuate, ameliorate, or maintain an existing disease or at least one symptom of the disease). The "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by PCSK9 expression, type of previous or concomitant therapy (if any), and other personal characteristics of the treated patient.

[0043] As used herein, a "prophylactically effective amount" refers to an amount of an RNAi agent sufficient to prevent or ameliorate a PCSK9-related disease or one or more symptoms thereof when administered to a subject who has not yet experienced or exhibited symptoms of the disease but may be susceptible to developing the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of subsequent disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity, as well as the patient's medical history, age, weight, family history, genetic makeup, previous or concurrent therapy (if any), and other personal characteristics of the treated patient.

[0044] A "therapeutically effective amount" or a "prophylactically effective amount" also includes that amount of an RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the present disclosure may be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0045] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from specific organs, organ parts, or fluids or cells within these organs. In some embodiments, samples may be derived from the liver (e.g., the entire liver or a portion of the liver, or certain cells of the liver (e.g., hepatocytes)). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma extracted from a subject. In some other embodiments, a "sample derived from a subject" refers to liver tissue (or a subcomponent thereof) derived from a subject.

[0046] In one aspect, the present disclosure provides a double-stranded RNAi agent for reducing PCSK9 expression, comprising any one selected from the following double-stranded oligonucleotides in which the sense strand and the antisense strand match: (1) a double-stranded oligonucleotide, the sense strand of which has the sequence set forth in SEQ ID NO. 1 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence set forth in SEQ ID NO. 13 or a fragment thereof, or a modified version of said sequence or said fragment; (2) a double-stranded oligonucleotide, the sense strand of which has the sequence set forth in SEQ ID NO. 2 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence set forth in SEQ ID NO. 14 or a fragment thereof, or a modified version of said sequence or said fragment; (3) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 3 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 15 or a fragment thereof, or a modified version of said sequence or said fragment; (4) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 4 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 16 or a fragment thereof, or a modified version of said sequence or said fragment; (5) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified version of said sequence or said fragment; (6) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 6 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 18 or a fragment thereof, or a modified version of said sequence or said fragment; (7) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 7 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 19 or a fragment thereof, or a modified version of said sequence or said fragment; (8) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified version of said sequence or said fragment; (9) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 9 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 21 or a fragment thereof, or a modified version of said sequence or said fragment; (10) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 10 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 22 or a fragment thereof, or a modified version of said sequence or said fragment; (11) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 11 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand of which has the sequence shown in SEQ ID NO. 23 or a fragment thereof, or a modified version of said sequence or said fragment; (12) A double-stranded oligonucleotide, wherein the sense strand has the sequence set forth in SEQ ID NO. 12 or a fragment thereof, or a modified sequence of said sequence or said fragment, and the antisense strand has the sequence set forth in SEQ ID NO. 24 or a fragment thereof, or a modified sequence of said sequence or said fragment.

[0047] In some embodiments, all nucleotides in the sense and antisense strands are modified nucleotides.

[0048] In some embodiments, the double-stranded RNAi agent is an RNAi agent used to inhibit PCSK9 gene expression.

[0049] In some embodiments, the sense strand differs from any one of SEQ ID NOs. 1-12 by 1-3 nucleotides.

[0050] In some embodiments, the antisense strand differs from any one of SEQ ID NOs. 13-24 by 1-3 nucleotides.

[0051] In some embodiments, the at least one modified nucleotide is selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base-containing nucleotide, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a thiophosphate-containing nucleotide, a methylphosphate-containing nucleotide, a 5'-phosphate-containing nucleotide, and a 5'-phosphate analog-containing nucleotide.

[0052] In some embodiments, at least one strand comprises a 3' overhang of at least one nucleotide.

[0053] In some embodiments, at least one strand comprises a 3' overhang of at least two nucleotides.

[0054] In some embodiments, the double-stranded region has a length of 15 to 30 nucleotide pairs.

[0055] In some embodiments, the double-stranded region has a length of 17 to 25 nucleotide pairs.

[0056] In some embodiments, the double-stranded region has a length of 19 to 23 nucleotide pairs.

[0057] In some embodiments, the double-stranded region has a length of 21 nucleotide pairs.

[0058] In some embodiments, each strand has between 15 and 30 nucleotides.

[0059] In some embodiments, each strand has between 19 and 25 nucleotides.

[0060] In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0061] In some embodiments, the modified form of all nucleotides in the sense and antisense strands is a chemical modification of the 2' position of the ribose of the nucleotide.

[0062] In some embodiments, the chemical modification at the 2'-position of the ribose of the nucleotide is any one or a combination of several groups selected from a 2'-methoxy group, a 2'-methoxyethyl group, a 2'-fluoro group, a 2'-benzyloxy group, a 2'-methylcarbonylamino group, and a 2'-pyridylmethoxy group.

[0063] In some embodiments, the chemical modification at the 2' position of the ribose of each nucleotide is selected from a combination of a 2'-methoxy group and a 2'-fluoro group.

[0064] In some embodiments, the chemical modification at the 2' position of the ribose of each nucleotide is selected from alternating combinations of 2'-methoxy and 2'-fluoro groups.

[0065] In some embodiments, the chemical modification of the 2'-position of the ribose of each nucleotide is 2'-fluoro modified at all odd-numbered positions in the sense strand, 2'-methoxy modified at all even-numbered positions in the sense strand, 2'-methoxy modified at all odd-numbered positions in the antisense strand, and 2'-fluoro modified at all even-numbered positions in the antisense strand.

[0066] In some embodiments, the nucleotide monomers are linked by 3',5'-phosphodiester bonds.

[0067] In some embodiments, the nucleotide monomers are linked together by thiolated modified 3',5'-phosphodiester bonds.

[0068] In some embodiments, the oligonucleotide comprises: The antisense strand is modified with any one of the following modifications A, B, and C, and the sense strand is modified with the following modifications a or b: TIFF2025541705000003.tif138168 (In the above table, 2'-OMe represents a 2'-methoxy group, 2'-F represents a 2'-fluoro group, and PS represents a thiophosphate backbone.) If modification A is used on the antisense strand, modification form a is used on the sense strand, When modification B is used in the antisense strand, modification form a is used in the sense strand, When modification C is used in the antisense strand, modification form a is used in the sense strand, If modification B is used on the antisense strand, modification form b is used on the sense strand, When modification C is used in the antisense strand, modification form b is used in the sense strand.

[0069] In some embodiments, positions 2 to 8 from the 5' end of the antisense strand are modified with a group selected from UNA, GNA, and DNA, where the structures of UNA and GNA are as follows: [ka] wherein the base is selected from adenine, guanine, cytosine, thymine, and uracil.

[0070] In some embodiments, the phosphorylation of the 5' carbon atom of the glycoside of the 5'-terminal nucleotide of the modified antisense strand may include, but is not limited to, the following 5'-position phosphorylation groups: 5'-vinylphosphonate (5'-E-VP), 5'-methylphosphonate (5'-MP), 5'-C-methylphosphate, 5'-thiophosphate (5'-PS), and 5'-phosphate (5'-P). [ka] (wherein R is hydrogen, a hydroxyl group, an amine group, C 1-4 Alkyl group, aryl group, C 1~4 Alkoxy group, C 1~4 alkylcarbonylamino group or halogen, The base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

[0071] In some embodiments, the 3',5'-phosphodiester bonds linking the nucleotide monomers at the ends of the sequence are modified with thiol groups to form chirally pure 3',5'-phosphorothiodiester bonds, the 5' ends of the sense strand and antisense strand contain 1 to 3 thiol groups, and the 3' end of the antisense strand contains 1 to 3 thiol groups.

[0072] The double-stranded RNA (dsRNA) agent of the present disclosure may optionally be conjugated to one or more ligands. The ligand may be attached to the 3'-end, 5'-end, or both ends of the sense strand, the antisense strand, or both strands. For example, the ligand may be conjugated to the sense strand. In a preferred embodiment, the ligand is attached to the 3'-end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand.

[0073] In another aspect, the present disclosure provides a conjugate (also referred to as a conjugate) for reducing PCSK9 expression, comprising the above-described double-stranded RNAi agent and a ligand conjugated thereto.

[0074] In some embodiments, the ligand is conjugated to the 3' or 5' end of the sense strand of the oligonucleotide.

[0075] In some embodiments, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0076] In some embodiments, the ligand is represented by the following structural formula: [ka] (wherein X is hydrogen, a hydroxyl protecting group including an acetyl group, a benzoyl group, and an isobutyryl group, or H; Y is an amine protecting group selected from the group consisting of a formyl group, an acetyl group, a propionyl group, an n-butyryl group, and an isobutyryl group, or H; n is an integer of 0 to 20; and q, r, and s are each independently an integer of 1 to 7.)

[0077] In some embodiments, the ligand is represented by the following structural formula: [ka]

[0078] In some embodiments, the ligand is represented by the following structural formula: [ka] wherein X is oxygen, nitrogen, or sulfur; Y is an alkyl group or an aryl group; R1 is oxygen or sulfur; R2 is hydrogen, amino group, C 1~4 Alkyl group, aryl group, C 1~4 is an alkoxy group or a halogen; A is -(CH2) a -, -(CH2CH2O) b -, -((CH2) c NHCO) d - or -((CH2) c CONH) d - (wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5), B is -(CH2) e- (wherein e is an integer from 0 to 7), L is -CONH- or -NHCO-; X1 is -(CH2) f -or-(CH2CH2O) f CH2- (wherein f is an integer of 1 to 5), X2 is -(CH2) g - (where g is an integer from 1 to 6), Y1 is 0 or 1; Y2 is 0, 1 or 2; Y3 is 1, 2 or 3; m is an integer from 0 to 4, n is an integer from 0 to 4.

[0079] In some embodiments, the ligand is represented by the following: G4, G5, G6, or G7. [ka]

[0080] In some embodiments, the conjugate (also called a conjugate) has the structure shown below: [ka]

[0081] In some embodiments, the double-stranded RNAi agent comprises any one selected from the following double-stranded oligonucleotides having matched sense and antisense strands and conjugated to ligands G4, G5, G6, or G7: (1) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 337 and the antisense strand of which has the sequence shown in SEQ ID NO. 427; (2) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 337 and the antisense strand of which has the sequence shown in SEQ ID NO. 428; (3) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 342 and the antisense strand of which has the sequence shown in SEQ ID NO. 381; (4) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 342 and the antisense strand of which has the sequence shown in SEQ ID NO. 430; (5) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 342 and the antisense strand of which has the sequence shown in SEQ ID NO. 431; (6) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 347 and the antisense strand of which has the sequence shown in SEQ ID NO. 384; (7) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 347 and the antisense strand of which has the sequence shown in SEQ ID NO. 437; (8) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 347 and the antisense strand has the sequence shown in SEQ ID NO. 438; (9) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 348 and the antisense strand has the sequence shown in SEQ ID NO. 385; (10) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 352 and the antisense strand has the sequence shown in SEQ ID NO. 448; (11) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO. 390; (12) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO. 448; (14) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO. 393; (15) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO. 446; (16) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO. 447.

[0082] In some embodiments, the double-stranded RNAi agent comprises any one selected from the following double-stranded oligonucleotides having matched sense and antisense strands and conjugated to ligands G4, G5, G6, or G7: (1) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 352 and the antisense strand of which has the sequence shown in SEQ ID NO. 448; (2) a double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 353 and the antisense strand of which has the sequence shown in SEQ ID NO. 390; (3) A double-stranded oligonucleotide, the sense strand of which has the sequence shown in SEQ ID NO. 353 and the antisense strand of which has the sequence shown in SEQ ID NO. 448.

[0083] In some embodiments, the double-stranded RNAi agent comprises a double-stranded oligonucleotide having a sense strand matched to SEQ ID NO. 353 and an antisense strand matched to SEQ ID NO. 448, and conjugated to ligand G5.

[0084] In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-described double-stranded RNAi agent or a conjugate thereof and a pharmaceutically acceptable carrier.

[0085] In some embodiments, the present specification provides a pharmaceutical composition comprising the RNAi described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the RNAi can be used to treat diseases or conditions associated with PCSK9 gene expression or activity, such as lipid disorders. Such pharmaceutical compositions are formulated based on a delivery model. One example is a systemic administration composition formulated for parenteral delivery, such as intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, such as by injection into the brain, such as by continuous pump infusion.

[0086] Pharmaceutical compositions comprising the RNAi agents of the present disclosure can be, for example, solutions with or without buffers, or compositions comprising a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micelle formulations, emulsions, and gene therapy vectors.

[0087] In the method of the present disclosure, the RNAi agent may be administered as a solution.The free RNAi agent may be administered as a non-buffered solution, such as saline or water.Alternatively, the free siRNA may be administered as a suitable buffer solution.This buffer solution includes acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.In a preferred embodiment, this buffer solution is phosphate-buffered saline (PBS).The pH and osmolality of the buffer containing the RNAi agent can be adjusted to be suitable for administration to a subject.

[0088] In some embodiments, the buffer solution further comprises a reagent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, such as the physiological value of human plasma. Solutes that can be added to the buffer solution to control osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the osmolality of the solution is a salt. In some embodiments, the reagent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0089] The pharmaceutical composition of the present disclosure may be administered at a dose sufficient to inhibit PCSK9 gene expression. Typically, a suitable dose of the RNAi of the present disclosure is in the range of about 0.001 to 200.0 mg / kg of subject body weight per day, usually in the range of about 1 to 50 mg / kg of subject body weight per day. For example, the RNAi agent (e.g., dsRNA) may be administered at a single dose of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.23, 3.24, 3.25, 3.26, 3.27, 3.28, 3.29, 3.33, 3.34, 3.35, 3.36, 3.37, 3.38, 3.39, 3.39, 3.40, 3.41, 3.42, 3.43, 3.44 .4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19 0.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / kg.

[0090] The pharmaceutical composition may be administered once a day, or the RNAi may be administered in two or more subdoses at appropriate intervals throughout the day, or by continuous infusion or delivery of a controlled-release formulation. In this case, the amount of RNAi contained in each subdose must be correspondingly small to achieve the total daily dose. The dosage unit may also be formulated to be delivered over several days, for example, using a conventional sustained-release formulation that sustains the release of RNAi over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, and can therefore be used with the drugs of the present disclosure. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0091] In other embodiments, a single dose of the pharmaceutical composition can be administered over an extended period of time such that the interval between subsequent doses is 3, 4, or 5 days or less, or 1, 2, 3, or 4 weeks or less. In some embodiments of the present disclosure, the pharmaceutical composition of the present disclosure is administered in a single dose once per week. In other embodiments of the present disclosure, the pharmaceutical composition of the present disclosure is administered in a single dose once per two months.

[0092] Those skilled in the art will appreciate that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other pre-existing conditions, may affect the dosage and duration required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective amount and in vivo half-life of each RNAi included in this disclosure can be estimated based on in vivo experiments using routine methods or appropriate animal models.

[0093] The pharmaceutical compositions of the present disclosure may be administered by a number of routes, depending on the local or systemic treatment and the area to be treated. Administration may be topical (e.g., using a skin patch), pulmonary (e.g., inhalation or insufflation of a powder or aerosol with a nebulizer), intratracheal, intranasal, epidermal and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subdermal administration such as by implant, or intracranial administration such as intracerebral parenchyma, intrathecal, or intraventricular administration.

[0094] The RNAi used in the compositions and methods of the present disclosure can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one or more bilayers). Liposomes include unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi composition. The lipid-soluble material typically separates the aqueous interior from an aqueous exterior that does not contain the RNAi composition (although in some cases may contain it). Liposomes are useful for transporting active ingredients and delivering them to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. Upon fusion of the liposome with the cell, the internal aqueous contents containing the RNAi are delivered to the cell, where the RNAi can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes may also be specifically targeted, for example, to direct RNAi to particular cell types.

[0095] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid components of the liposomes are dissolved in a detergent to form micelles with the lipid components. For example, the lipid components may be amphipathic cationic lipids or lipid conjugates. The detergent may have a high critical micelle concentration or may be non-ionic. Examples of detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent formulation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome formulation of the RNAi agent.

[0096] RNAi, such as the dsRNA of this disclosure, can be fully packaged in a lipid formulation (eg, an LNP or other nucleic acid-lipid particle).

[0097] The term "LNP" used herein refers to stable nucleic acid-lipid particles.LNP comprises cationic lipid, non-cationic lipid, and lipid that prevents particle aggregation (for example, PEG-lipid conjugate).LNP has a long circulation life after intravenous (iv) injection, and accumulates at distant sites (for example, sites physically separated from the administration site), making it extremely useful for synthetic applications.

[0098] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) is in the range of about 1:1 to 50:1, about 1:1 to 25:1, about 3:1 to 15:1, about 4:1 to 10:1, about 5:1 to 9:1, or about 6:1 to 9:1.

[0099] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

[0100] In some preferred embodiments, the cationic lipid is a compound having the structure represented by the following formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as YK-009 (see Patent CN114044741B) having the structure represented by the following formula (II): [ka] (Wherein G1 is C 1~6 is an alkylene group, and G2 is C 2~8 alkylene group, G3 is C 1~3 is an alkylene group, and L1 is C 6~15 A straight chain alkyl group, L2 is C 12~25 It is a branched alkyl group.

[0101] In some preferred embodiments, the cationic lipid is a compound represented by the following formula (II) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as YK-401 having the following structure: (II-I) and YK-402 having the following structure: (II-II) (see Patent CN115784921B). [ka] (Wherein G1 is C 2~8 is an alkylene group, and G2 is C 2~8 an alkylene group, L1 is C(O)O- or -OC(O)-, L2 is C(O)O- or -OC(O)-, and R1 is C 6~25 is a straight or branched alkyl group, and R2 is C 6~25 is a straight-chain or branched alkyl group, G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, and L is (CH2)2- or -(CH2)3- or -(CH2)4-.

[0102] In some preferred embodiments, the cationic lipid is a compound represented by the following formula (III) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as YK-201 having the following structure: (III-I) and YK-202 having the following structure: (III-II) (see Patent CN115677518B). [ka] (Wherein G1 is C 1~6 is an alkylene group, and G2 is C 2~8 is an alkylene group, and R1 is C 6~20 is a straight or branched alkyl group, and R2 is C 12~25 is a branched alkyl group, and G3 is HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CHO(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-.

[0103] In some preferred embodiments, the cationic lipid is a compound represented by the following formula (IV) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as YK-305 having the structure of formula (IV-I) and YK-310 having the structure of formula (IV-II) (see Patent CN115745820B). [ka] (Wherein G1 is C 1~8 is an alkylene group, and G2 is C 2~8 is an alkylene group, and R1 is C 6~25 is a straight or branched alkyl group, and R2 is C 12~25 is a straight-chain or branched alkyl group, and G3 is HO(CH2)2N(R3)CH2CH(OH)CH2- (wherein R3 is -CH3, -CH2CH3, or -CH2CH2OH).

[0104] In some preferred embodiments, the cationic lipid is a compound represented by the following formula (V) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as ALC0315 (see Patent CN108368028B) having the following formula (VI): [ka] (In the formula, G 1 and G 2 are each independently unsubstituted C6 to C 10 is an alkylene group, and G 3 is unsubstituted C1~C 12 is an alkylene group, and R1 and R2 are each independently C6 to C 24 Alkyl group or C6-C 24 is an alkenyl group, R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1~C 12 is a hydrocarbon group, R 5 is H or a C1-C6 hydrocarbon group.

[0105] In some preferred embodiments, the cationic lipid is a compound represented by the following formula (VI) or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, such as SM102 (see Patent CN110520409A) having the following structure: [ka] (Wherein R4 is —(CH2) n Q or -(CH2) n CHQR, where Q is -OR, -OH, or -O(CH2) nand a heterocycle, wherein n is 1, 2, or 3; and each R is independently selected from the group consisting of N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(H)(R), -N(R)C(S)N(R), -N(H)C(S)N(H)(R), -N(R)S(O)R and a heterocycle; 1~3 Alkyl group, C 2~3 Alkenyl group, (CH2) q and H, each q is independently selected from 1, 2, and 3; and each R* is independently selected from C 1~12 Alkyl group or C 2~12 alkenyl groups, and each X is independently selected from the group consisting of F, Cl, Br, and I.

[0106] In some preferred embodiments, the cationic lipid is a compound represented by formula (VII) below, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof (see CN102625696B, DLIN-MC3-DMA): [ka]

[0107] In some more preferred embodiments, the cationic lipids include YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0108] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0109] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0110] In some preferred embodiments, the molar ratio of the cationic lipid:the neutral lipid:the structural lipid:the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0111] In some preferred embodiments, the molar ratio of the cationic lipid:the neutral lipid:the structural lipid:the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0112] In some more preferred embodiments, the molar ratio of the cationic lipid:the neutral lipid:the structured lipid:the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0113] In some preferred embodiments, the neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0114] In some more preferred embodiments, the neutral lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16).0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerin) (DOPG) sodium salt, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl- ... and at least one selected from the group consisting of 1-stearoyl-2-oleoyl-stearylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0115] In some more preferred embodiments, the neutral lipid is DOPE and / or DSPC.

[0116] In some preferred embodiments, the structured lipid is one or more selected from cholesterol, a non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and a corticosteroid.

[0117] In some more preferred embodiments, the structured lipid is cholesterol.

[0118] In some preferred embodiments, the polymer-bound lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0119] In some more preferred embodiments, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0120] The pharmaceutical compositions of the present disclosure include, but are not limited to, solution, emulsion and liposome-containing preparation.These compositions can be prepared from various components such as preformed liquid, self-emulsifying solid and self-emulsifying semisolid.Particularly preferred is the preparation that targets the liver when treating liver damage (for example, liver cancer).

[0121] The pharmaceutical formulations of the present disclosure, which may conveniently be in unit dosage form, can be prepared according to conventional methods well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredients with their pharmaceutical carriers or excipients. Generally, these formulations are prepared by uniformly and finely combining the active ingredients with liquid carriers or finely dispersed solid carriers, or both, which can then be further shaped into a product, if necessary.

[0122] The compositions of the present disclosure may be prepared into any possible dosage form. Dosage forms include, but are not limited to, tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions of the present disclosure may also be prepared as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain agents that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers.

[0123] Certain compositions of the present disclosure further include a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analog that is inert (i.e., not biologically active by itself) but is considered a nucleic acid in vivo because it degrades biologically active nucleic acids or promotes their removal from the circulation, thereby reducing their bioavailability. The combined use of nucleic acids and a carrier compound (usually in excess of the latter) can significantly reduce the amount of nucleic acid recovered in the liver, kidney, or other extracorporeal circulation reservoir, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, when combined with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid, the recovery of partially thiophosphorylated dsRNA in liver tissue can be reduced.

[0124] In contrast to a carrier compound, a "drug carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Such excipients may be liquid or solid and are selected to provide the desired volume, viscosity, etc., when combined with the nucleic acids and other components of a particular pharmaceutical composition, with reference to the desired means of administration. Typical drug carriers include, but are not limited to, binders (e.g., pregelatinized cornstarch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, and the like), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate, and the like), lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, cornstarch, polyethylene glycol, sodium benzoate, sodium acetate, and the like), disintegrants (e.g., starch, sodium starch glycolate, and the like), and wetting agents (e.g., sodium lauryl sulfate, and the like).

[0125] Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not toxically react with nucleic acids can also be used to prepare the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, or polyvinylpyrrolidone.

[0126] Formulations for topical administration of nucleic acids may include solutions of nucleic acids in common solvents such as alcohol, sterile or non-sterile aqueous solutions, non-aqueous solutions, or liquid or solid oil matrices. These solutions may include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not toxically react with nucleic acids can be used.

[0127] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, or polyvinylpyrrolidone.

[0128] The dosage forms, carrier compounds, drug carriers, excipients, etc. of the above compositions are described in U.S. Patent No. 10,125,369 B2, the contents of which are incorporated herein by reference.

[0129] The present disclosure also provides methods for treating or preventing diseases or conditions that can be regulated by downregulating PCSK9 gene expression. For example, the RNAi agents described herein can be used to treat lipidemias, such as hyperlipidemia, and other forms of dyslipidemia, such as hypercholesterolemia and hypertriglyceridemia, as well as pathological conditions associated with these disorders, such as heart and circulatory system diseases. Other diseases or conditions that can be regulated by downregulating PCSK9 gene expression include, but are not limited to, lysosomal storage disorders, such as Niemann-Pick disease, Tay-Sachs disease, lysosomal acid lipase deficiency, and Gaucher disease. The RNAi agents described herein can be used to treat cardiovascular diseases such as coronary artery disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), transient ischemic attack (TIA), angina pectoris (stable or unstable), atrial fibrillation, arrhythmia, valvular disease, and / or congestive heart failure, or any other condition. These methods include administering a therapeutically or prophylactically effective amount of an RNAi agent, conjugate, or composition of the present disclosure to a subject. In some embodiments, the method includes administering a therapeutically effective amount of PCSK9 siRNA to a patient with a heterozygous LDLR gene mutation.

[0130] The RNAi agent of the present disclosure can be administered to subject by any administration means known in the art.Administration means include but are not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal and any combination thereof.In a preferred embodiment, these agents are administered subcutaneously.

[0131] In other embodiments, the siRNA may be administered in combination with another therapeutic agent. The siRNA and the other therapeutic agent may be administered together in the same composition, e.g., parenterally, or the other therapeutic agent may be administered as part of a separate composition or by another method described herein.

[0132] Examples of other therapeutic agents include drugs known to treat dyslipidemia, such as hypercholesterolemia, atherosclerosis, dyslipidemia, or cardiovascular and cerebrovascular diseases. For example, other therapeutic agents for treating hyperlipidemia are selected from fibrates, statins, bile acid sequestrants, and nicotinic acid drugs. Other therapeutic agents for treating cardiovascular and cerebrovascular diseases are selected from angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril, benazepril, perindopril, etc.), angiotensin II receptor antagonists (e.g., losartan, losartan / hydrochlorothiazide, valsartan, valsartan / hydrochlorothiazide, telmisartan, telmisartan / tanhydrochlorothiazide, olmesartan medoxomil, etc.), and beta-receptor blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0133] In some embodiments, the RNAi agent is administered to the patient before the other therapeutic agent is administered to the patient (or vice versa). In some other embodiments, the RNAi agent and the other therapeutic agent are administered simultaneously.

[0134] In another aspect, the disclosure provides the use of the double-stranded RNAi agent, conjugate, or composition in the manufacture of a medicament for treating a PCSK9-related disease.

[0135] In another aspect, the disclosure provides the use of the double-stranded RNAi agent, conjugate, or composition in the manufacture of a medicament for treating hypercholesterolemia, atherosclerosis, dyslipidemia, or cardiovascular and cerebrovascular diseases.

[0136] In some embodiments, the PCSK9-associated disease is selected from hypercholesterolemia, atherosclerosis, dyslipidemia, cardiovascular and cerebrovascular disease.

[0137] The following examples are used to illustrate the present disclosure, but are not intended to limit the scope of the present disclosure. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0138] Nucleotide abbreviations used herein are as follows: A = adenosine 3'-phosphate Am = 2'-O-methoxyadenosine-3'-phosphate Ams = 2'-O-methoxyadenosine-3'-thiophosphate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-thiophosphate G = guanosine-3'-phosphate Gm = 2'-O-methoxyguanosine-3'-phosphate Gms = 2'-O-methoxyguanosine-3'-thiophosphate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-thiophosphate C=cytidine-3'-phosphate Cm = 2'-O-methoxycytidine-3'-phosphate Cms = 2'-O-methoxycytidine-3'-thiophosphate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-thiophosphate U = uridine-3'-phosphate Um = 2'-O-methoxyuridine-3'-phosphate ester Ums = 2'-O-methoxyuridine-3'-thiophosphate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-thiophosphate AmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyadenosine-3'-thiophosphate UmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyuridine-3'-thiophosphate Agna = Adenosine-glycol nucleic acid Cgna = cytidine-glycol nucleic acid Ggna = Guanosine-glycol nucleic acid Tgna = thymidine-glycol nucleic acid Ugna = Uridine-glycol nucleic acid [Example]

[0139] Example 1 Synthesis of Alternately Modified Small Interfering Oligonucleotides Eighty-four siRNA base sequences were designed based on the PCSK9 mRNA sequence. To improve the inhibitory efficacy and stability of the sequences, the base sequences were alternately modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) and then modified with thiol at the termini. The sense strand was modified with 2'-F at all odd-numbered positions and 2'-OMe at all even-numbered positions, while the antisense strand was modified with 2'-OMe at all odd-numbered positions and 2'-F at all even-numbered positions. Furthermore, the sense strand had two thiol modifications at its 5' end, and the antisense strand had two thiol modifications at its 5' and 3' ends, respectively. The alternatingly modified siRNA sequences are shown in Table 2.

[0140] 1. Synthesis of the Alternately Modified Sequence P92-si5 The basic sequence of the small interfering RNA with the sequence number P92-si5 in Table 2 is: Sense strand: 5'-AAGAUCCUGCAUGUCUUCCAU-3' (SEQ ID NO. 1), The antisense strand was 5'-AUGGAAGACAUGCAGGAUCUUGG-3' (SEQ ID NO. 13). The odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand were modified with 2'-F, and the other positions were modified with 2'-OMe. In addition, there were two thiol modifications at the 5' end of the sense strand and two thiol modifications at the 5' and 3' ends of the antisense strand.

[0141] Equipment and reagents: DNA / RNA automated synthesizer (Model No. 192P, Beijing Tsingke Biotech Co., Ltd.) Solid support: General-purpose support of cross-linked polystyrene beads (product number Primer support 5G Unylinker 350, Cytiva Co., Ltd.)

[0142] Preparation method: Nucleotide monomer solutions such as DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), ​​DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), and DMT-UF phosphoramidite monomer (Formula 8) were prepared using acetonitrile at a monomer concentration of 0.15 M. [ka]

[0143] It was prepared by the following steps. (1) Deprotection The DMT protecting group was removed using a 3% dichloroacetic acid-toluene solution as a deprotecting reagent, followed by washing with acetonitrile. (2) Coupling For an acetonitrile solution of each nucleotide monomer, coupling was carried out using 0.25 M 5-(ethylthio)-1H-tetrazole as an activating agent, followed by washing with acetonitrile. (3) Oxidation / sulfurization Oxidation: Oxidation was carried out using a 0.05 M solution of iodine in pyridine / water (90 / 10) as an oxidizing agent, followed by washing with acetonitrile. Sulfurization: Sulfurization was carried out using a pyridine solution of 3% xanthan hydride as a sulfurizing agent, followed by washing with acetonitrile. (4) Protection of hydroxyl groups The hydroxyl groups were protected using 10% acetic anhydride in tetrahydrofuran (Cap A) or tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (Cap B) as a protecting agent for the hydroxyl groups, followed by washing with acetonitrile. The above steps were repeated, cycling through a set sequence to obtain the full protected product. (5) The DMT protecting group of the last nucleotide was removed using a 3% dichloroacetic acid-toluene solution as a deprotection reagent, followed by washing with acetonitrile. (6) Aminolysis and purification The solid support was transferred to a reactor and concentrated aqueous ammonia (25-28%) was added. The aminolysis was maintained at 60°C for 12 hours, after which it was cooled to room temperature. The mixture was transferred to a filter press and washed with a mixture of purified water and ethanol. The combined filtrate was passed through a chromatography column, concentrated, and lyophilized to obtain the product. (7) Annealing The purified sense strand and antisense strand were mixed in a 1:1 ratio, heated to 95°C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double strand. Purity of P92-si5: 97.4%, Measured molecular weight: 14493.52

[0144] 2. Synthesis of other sequences Other sequences shown in Table 2 were synthesized according to the methods described above.

[0145] [Table 2] TIFF2025541705000020.tif247168TIFF2025541705000021.tif247168TIFF2025541705000022.tif247168TIFF2025541705000023.tif157168

[0146] Example 2 Inhibitory effect of alternately modified sequences on the PCSK9 gene The 2'-OMe and 2'-F alternating modified siRNA sequences synthesized in Example 1 were transfected into HELA cells via lipid nanoparticles (LNPs). The inhibitory effect of each sequence on the PCSK9 gene was measured using qPCR technology.

[0147] 1. Experimental Materials Sample: Alternately modified small interfering RNA sequence P92-si1-84 (synthesized in Example 1) shown in Table 2. Cell type: HeLa cell line HELA cells Drug solvent: enzyme-free sterile water, Gibco Opti-MEM.

[0148] 2. Experimental Method The inhibitory effect of the samples on the mRNA expression of the PCSK9 gene in HELA cell lines was measured using qRT-PCR.

[0149] 2.1 Cell culture Subcultured HELA cell lines were harvested. Logarithmically growing cells were cultured in 10% fetal bovine serum RPMI 1640 medium (supplemented with 100x penicillin and streptomycin at 10 μL / mL) in a 37°C, 5% CO2 incubator, with the medium replaced once daily. Cells were digested with 0.25% trypsin and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and new medium was added for subculture.

[0150] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a ratio of 1.5:98.5, and then mixed using a vortex mixer. Preparation of transfection reagent: 60 μL of siRNA solution diluted with Opti-MEM was added to 60 μL of transfection mixture at a 1:1 (v / v) ratio, vortexed, and then allowed to stand at room temperature for 15 minutes to obtain lipid nanoparticles (LNPs). 12.5 μL of the resulting LNPs were used for encapsulation efficiency measurements. Transfection reagent for blank control group: 60 μL of the prepared transfection mixture was added to 60 μL of Opti-MEM, mixed with a vortex mixer, and allowed to stand at room temperature for 15 minutes. The prepared transfection reagent was added to a 24-well cell culture plate (100 μL / well) so that the final siRNA concentration was 1 nM / well. 4 The mixture was mixed using the cross method and placed in an incubator at 37°C with 5% CO2 for 40 hours.

[0151] 2.3 PCSK9 mRNA measurement 1) RNA extraction a. The medium was removed from the 12-well plate. 0.5 mL of 1x PBS was added to each well to wash the cells, and the PBS was removed. 0.5 mL of TRIzol reagent was added to each well. The cells were completely lysed by pipetting, transferred to a 1.5 mL RNAse-free EP tube, and left at room temperature for 5 minutes. b. 0.1 mL of chloroform was added to each tube, which was then vigorously shaken for 15 seconds and allowed to stand at room temperature for 5 minutes. After centrifugation at 12,000 × g for 15 minutes at 4°C, 200 μL of the supernatant was transferred to a new EP tube. c) An equal volume of isopropanol was added. The liquid in the tube was gently mixed by inverting it and allowed to stand at -20°C for 10 minutes. After centrifugation at 12,000 x g for 15 minutes at 4°C, the supernatant was discarded. d. 0.5 mL of 75% ethanol was added to gently wash the RNA precipitate. After centrifugation at 12,000 × g for 5 minutes at 4°C, the supernatant was removed. This washing was repeated once. After centrifugation at 12,000 × g for 1 minute at 4°C, the residual ethanol was removed using a micropipette tip. e. The remaining ethanol was dried at room temperature for 2-3 minutes, and then dissolved by adding 40 μL of RNase-free ddH2O. 2) Measurement of RNA concentration RNA concentrations were measured using a NanoDrop. 2 μL of RNA sample was used for each measurement, with 2 μL of RNase-free ddH2O as a control. Sample concentrations were recorded. 3) Quantitative measurement of PCSK9 mRNA The remaining components except for the primers and template were added to a 15 mL centrifuge tube at 7.5 x 84 = 630 parts and marked as A. Eighty-four 1.5 mL EP tubes were marked. Seven tubes each (77 μL of A and 420 ng of total RNA) were added and mixed (marked as B). The upstream and downstream primers of the internal control gene GAPDH and the target gene PCSK9 were mixed (marked as C). 11 μL of B+1 μL of C was added to each well of the PCR plate, covered with sealing film, centrifuged at 3000 rpm for 1 minute, and transferred to the device. *This step was performed on ice to maintain cold conditions. The plate was placed in a qPCR machine and run according to the following program: Reverse transcription: 55°C, 15 minutes. Heat denaturation: 95°C, 30 seconds. Cycle reaction: 95°C, 10 seconds, 60°C, 35 seconds, 40 cycles. Melting curve: 95°C, 15 seconds. 60°C, 60 seconds. 95°C, 15 seconds. The run time was approximately 2 hours. The experimental results were analyzed and 2-ΔΔCt was calculated.

[0152] 2.4 Data Processing The PCSK9 mRNA expression rate (%) was calculated as follows. Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) x 100% Inhibition rate of PCSK9 gene expression = 1 - expression rate (%).

[0153] 3. Experimental Results The inhibition rate in this example is the average value of four experiments. The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4 to 6. Experimental results showed that alternating 2'-OMe and 2'-F modified sequences, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83 (specific sequences are shown in Table 3), had significant inhibitory effects on PCSK9 mRNA expression in HELA cells, with inhibition rates exceeding 50%. Among them, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 had inhibition rates of over 70%. The other sequences had relatively low inhibitory effects on PCSK9 mRNA expression, with the inhibition rates all being less than 50%.

[0154] [Table 3]

[0155] The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4 to 6. (1) Of the 84 sequences designed, 12 sequences, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83, exhibited significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among them, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exhibited inhibition rates exceeding 70%.

[0156] [Table 4]

[0157] Table 4 shows that a total of 12 alternating modified sequences, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83 (the specific sequences are shown in Table 3), have significant inhibitory effects on PCSK9 mRNA expression, with the inhibition rates all exceeding 50%. Among them, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 had inhibition rates exceeding 70% (Figure 1). These 12 sequences can be used as candidate sequences.

[0158] (2) The 26 sequences had inhibitory rates of 30% to 50% against the PCSK9 gene. For example, the inhibitory rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0159] [Table 5]

[0160] The 26 sequences shown in Table 5 had low inhibitory effects on the PCSK9 gene, with the inhibition rates being less than 50% and ranging from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively (Figure 2).

[0161] (3) Forty-six sequences showed an inhibitory rate of 30% or less against the PCSK9 gene. For example, the inhibitory rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0162] [Table 6] TIFF2025541705000028.tif19168

[0163] The 46 sequences listed in Table 6 all had very low inhibitory effects on PCSK9 mRNA expression, with inhibition rates of less than 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively (Figures 3 and 4).

[0164] (4) siRNAs with similar sequences showed very different activities. For example, P92-si5 showed a significantly improved inhibitory rate of 61.8% compared with P92-si4.

[0165] Table 7 shows a comparison of the inhibitory effects of siRNAs with similar sequences on PCSK9 mRNA.

[0166] [Table 7]

[0167] Table 7 shows that siRNAs with similar sequences have significantly different inhibitory effects on PCSK9 mRNA expression. Base sequence 4 differed from base sequence 5 by only one base at the end. In the sense strand, base sequence 4 had one extra C at the 5' end, and base sequence 5 had one extra U at the 3' end, with the remaining bases being identical. In the antisense strand, base sequence 4 had one extra U at the 3' end, and base sequence 5 had one extra A at the 5' end, with the remaining bases being identical. However, the alternately modified sequence P92-si5 showed a significant improvement in inhibition, with a 61.8% increase over P92-si4.

[0168] Base sequence 50 differed from base sequence 51 by only six bases at the end. In the sense strand, the 5' end of base sequence 50 was GAUUAA, and the 3' end of base sequence 51 was CUGGAU, with the remaining bases identical. In the antisense strand, the 3' end of base sequence 50 was AAUCAG, and the 5' end of base sequence 51 was AUCCAG, with the remaining bases identical. However, the alternately modified sequence P92-si51 showed a significantly improved inhibition rate, 46.8% higher than that of P92-si50.

[0169] Base sequence 2 differed from base sequence 3 by only six bases at the end. In the sense strand, the 5' end of base sequence 2 was AUACCU, the 3' end was UGUCUU, and the remaining bases were identical. In the antisense strand, the 3' end of base sequence 2 was GUAUCC, and the 5' end of base sequence 3 was AAGACA, and the remaining bases were identical. However, the alternating modified sequence P92-si3 showed a significant improvement in inhibition, with a 45.9% increase over P92-si2.

[0170] Base sequence 9 differed from base sequence 10 by only two bases at the end. In the sense strand, the 5' end of base sequence 9 was GG, and the 3' end of base sequence 10 was AG, with the remaining bases being identical. In the antisense strand, the 3' end of base sequence 9 was UA, and the 5' end of base sequence 10 was CU, with the remaining bases being identical. However, the alternately modified sequence P92-si9 showed a significantly improved inhibition rate of 53.1% compared to P92-si10.

[0171] Base sequence 21 differed from base sequence 22 only by two bases at the end. In the sense strand, the 5' end of base sequence 21 was CA, and the 3' end of base sequence 22 was GA, with the remaining bases being identical. In the antisense strand, the 3' end of base sequence 21 was CA, and the 5' end of base sequence 22 was UC, with the remaining bases being identical. However, the alternately modified sequence P92-si21 showed a significantly improved inhibition rate of 65.6% compared to P92-si22.

[0172] Base sequence 30 differed from base sequence 31 by only one base at the end. In the sense strand, base sequence 30 had an A at the 5' end and base sequence 31 had a G at the 3' end, with the remaining bases being identical. In the antisense strand, base sequence 30 had a G at the 3' end and base sequence 31 had a C at the 5' end, with the remaining bases being identical. However, the alternately modified sequence P92-si31 showed a significantly improved inhibition rate of 39.4% compared to P92-si30.

[0173] Base sequence 73 differed from base sequence 74 by only one base at the end. In the sense strand, base sequence 73 had a G at the 5' end, base sequence 74 had a G at the 3' end, and the remaining bases were identical. In the antisense strand, base sequence 73 had an A at the 3' end, and base sequence 74 had a C at the 5' end, and the remaining bases were identical. However, the alternately modified sequence P92-si73 showed a significantly improved inhibition rate of 62.2% compared to P92-si74.

[0174] Therefore, it is not easy to select sequences with significant inhibitory activity from the large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and much ingenuity is required.

[0175] summary: (1) Of the 84 sequences designed, 12 sequences, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83, exhibited significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among these, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exhibited inhibition rates exceeding 70%. (2) The 26 sequences had inhibitory rates of 30% to 50% against the PCSK9 gene. For example, the inhibitory rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively. (3) Forty-six sequences showed an inhibitory rate of 30% or less against the PCSK9 gene. For example, the inhibitory rates of P92-si1 and P92-si20 were 9.2% and 6.0%, respectively. (4) Even siRNAs with similar sequences showed very different activities. For example, P92-si5 had a significantly improved inhibitory rate of 61.8% compared to P92-si4. Therefore, selecting sequences with significant inhibitory activity from the large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence is not easy and requires considerable ingenuity.

[0176] Example 3: Inhibitory effect of unmodified sequence on PCSK9 gene In this example, several unmodified sequences corresponding to the modified sequences in Example 2 were synthesized and transfected into HELA cells via lipid nanoparticles (LNPs). The inhibitory effect of each unmodified sequence on the PCSK9 gene was measured using qPCR technology, and unmodified siRNA sequences with good inhibitory effects were selected.

[0177] 1. Experimental Materials Samples: Unmodified small interfering RNA sequences shown in Table 8. All sequences were synthesized according to the method in Example 1.

[0178] [Table 8] TIFF2025541705000031.tif222168

[0179] Cell type: HeLa cell line HELA cells Drug solvent: enzyme-free sterile water, Gibco Opti-MEM.

[0180] 2. Experimental Method See Example 2. 3. Experimental Results The inhibition rates of each unmodified sequence on PCSK9 mRNA expression are shown in Tables 9 and 10. The unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83 all had significant inhibitory effects on PCSK9 mRNA expression in HELA cells, with inhibition rates of over 50%. Among them, the inhibition rates of si5, si81, si82, si3, and si8 exceeded 60%.

[0181] (1) Twelve unmodified sequences, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, had significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among them, the inhibition rates of si5, si81, si82, si3, and si8 exceeded 60%.

[0182] [Table 9]

[0183] The unmodified sequences shown in Table 9, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, had significant inhibitory effects on PCSK9 mRNA expression, with inhibition rates of 50% or higher. Among them, the inhibition rates of si5, si81, si82, si3, and si8 exceeded 60% (Figure 5). These 12 sequences can be used as candidate sequences.

[0184] (2) The inhibition rates of other unmodified sequences against the PCSK9 gene were all less than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2%, respectively.

[0185] [Table 10]

[0186] Each of the sequences in Table 10 had a very low inhibitory effect on PCSK9 mRNA expression, with the inhibition rate being less than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2%, respectively (Figure 6).

[0187] (3) siRNAs with similar sequences showed very different activities. For example, base sequence 5 showed a significantly improved inhibitory rate of 51.2% compared with base sequence 4. Table 11 shows a comparison of the inhibitory effects of siRNAs with similar sequences on PCSK9 mRNA.

[0188] [Table 11]

[0189] Table 11 shows that siRNAs with similar sequences have very different inhibitory effects on PCSK9 mRNA expression.

[0190] Base sequence 4 differed from base sequence 5 by only one base at the end. In the sense strand, base sequence 4 had one extra C at the 5' end and base sequence 5 had one extra U at the 3' end, with the remaining bases being identical. In the antisense strand, base sequence 4 had one extra U at the 3' end and base sequence 5 had one extra A at the 5' end, with the remaining bases being identical. However, base sequence 5 showed a significant improvement in inhibition, with a 51.2% increase over base sequence 4.

[0191] Base sequence 50 differed from base sequence 51 by only six bases at the end. In the sense strand, the 5' end of base sequence 50 was GAUUAA, and the 3' end of base sequence 51 was CUGGAU, with the remaining bases being identical. In the antisense strand, the 3' end of base sequence 50 was AAUCAG, and the 5' end of base sequence 51 was AUCCAG, with the remaining bases being identical. However, base sequence 51 showed a significantly improved inhibition rate of 42.6% compared to base sequence 50.

[0192] Base sequence 2 differed from base sequence 3 by only six bases at the end. In the sense strand, the 5' end of base sequence 2 was AUACCU, and the 3' end of base sequence 3 was UGUCUU, with the remaining bases being identical. In the antisense strand, the 3' end of base sequence 2 was GUAUCC, and the 5' end of base sequence 3 was AAGACA, with the remaining bases being identical. However, base sequence 3 showed a significantly improved inhibition rate of 42.7% over base sequence 2.

[0193] Base sequence 9 differed from base sequence 10 by only two bases at the end. In the sense strand, the 5' end of base sequence 9 was GG, and the 3' end of base sequence 10 was AG, with the remaining bases being identical. In the antisense strand, the 3' end of base sequence 9 was UA, and the 5' end of base sequence 10 was CU, with the remaining bases being identical. However, base sequence 9 showed a significantly improved inhibition rate of 41.2% compared to base sequence 10.

[0194] Base sequence 21 differed from base sequence 22 only in the two terminal bases. In the sense strand, the 5' end of base sequence 21 was CA, and the 3' end of base sequence 22 was GA, with the remaining bases being exactly the same. In the antisense strand, the 3' end of base sequence 21 was CA, and the 5' end of base sequence 22 was UC, with the remaining bases being exactly the same. However, base sequence 21 showed a significantly improved inhibition rate of 53.2% compared to base sequence 22.

[0195] Base sequence 30 differed from base sequence 31 by only one base at the end. In the sense strand, the 5' end of base sequence 30 was A, the 3' end of base sequence 31 was G, and the remaining bases were exactly the same. In the antisense strand, the 3' end of base sequence 30 was G, the 5' end of base sequence 31 was C, and the remaining bases were exactly the same. However, base sequence 31 showed a significantly improved inhibition rate of 32.5% compared to base sequence 30.

[0196] Base sequence 73 differed from base sequence 74 by only one base at the end. In the sense strand, base sequence 73 had a G at the 5' end and base sequence 74 had a G at the 3' end, with the remaining bases being identical. In the antisense strand, base sequence 73 had an A at the 3' end and base sequence 74 had a C at the 5' end, with the remaining bases being identical. However, base sequence 73 had a significantly improved inhibition rate of 50.2% compared to base sequence 74. Therefore, it is not easy to select sequences with significant inhibitory activity from the large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and much ingenuity is required.

[0197] (4) The effects of alternating modification on different sequences varied. For example, alternating modification of base sequence 5 significantly improved the inhibition rate (12.4%) compared with the unmodified sequence. However, alternating modification of base sequences 4, 22, and 52 did not significantly affect the inhibition rate compared with the unmodified sequence.

[0198] [Table 12]

[0199] Table 12 shows that alternating modifications to different sequences had various effects on the inhibitory effect on PCSK9 mRNA. For example, there were cases where the sequence obtained by alternating modification of base sequence 5 had a 12.4% higher inhibitory rate than the unmodified sequence, and there were cases where the sequence obtained by alternating modification of base sequence 82 had a 12.2% higher inhibitory rate than the unmodified sequence, resulting in a significant improvement in inhibitory rate. There were also cases where the sequences obtained by alternating modification of base sequences 4, 22, and 52 had almost no change in inhibitory rate compared to the unmodified sequence. Therefore, not all unmodified sequences can be improved in activity by alternating modifications; alternating modifications have different effects on the activity of different sequences.

[0200] summary: (1) Twelve unmodified sequences, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, had significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among them, the inhibition rates of si5, si81, si82, si3, and si8 exceeded 60%. (2) The inhibition rates of other unmodified sequences against the PCSK9 gene were less than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2%, respectively. (3) siRNAs with similar sequences showed very different activities. For example, base sequence 5 showed a significantly improved inhibitory rate of 51.2% compared with base sequence 4. (4) Alternative modification of different sequences had various effects on activity. For example, in one case, the sequence obtained by alternating modification of base sequence 5 showed a significant improvement in inhibition rate (12.4%) compared to the unmodified sequence. In another case, the sequences obtained by alternating modification of base sequences 4, 22, and 52 showed almost no change in inhibition rate compared to the unmodified sequence.

[0201] Example 4: Inhibitory effect of template-modified sequences on the PCSK9 gene In this example, the sequences selected in Example 3, i.e., a total of 12 sequences, unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, were modified using modification templates. Among these, DV25, DV26, DV27, DV28, DV29, DV30, and DV31 are new modification templates designed in this disclosure, and DV21 and DV22 are the disclosed Advanced ESC modification templates. 1. Experimental Materials Samples: Table 13 shows the sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73 and si83 in Example 3 modified with different templates (DV25, DV26, DV27, DV28, DV29, DV30, DV31, DV21 and DV22), and their corresponding alternately modified sequences P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73 and P92-si83 (synthesized in Example 1). The modification principle of the modified template of the present disclosure is as follows.

[0202] TIFF2025541705000036.tif153168

[0203] The siRNA-modified template, which used modification form A in the antisense strand and modification form a in the sense strand, was named DV25. The siRNA-modified template, which used modification form B in the antisense strand and modification form a in the sense strand, was named DV26. The siRNA-modified template, which used modification form C in the antisense strand and modification form a in the sense strand, was named DV27. The siRNA-modified template, which used modified form B in the antisense strand and modified form b in the sense strand, was named DV28. The siRNA-modified template, which used modification form C in the antisense strand and modification form b in the sense strand, was named DV29.

[0204] TIFF2025541705000037.tif99168

[0205] TIFF2025541705000038.tif94168

[0206] TIFF2025541705000039.tif80168

[0207] TIFF2025541705000040.tif81168

[0208] Details of each modified template sequence are shown in Table 13. The synthesis method for each sequence was the same as in Example 1.

[0209] [Table 13] TIFF2025541705000042.tif247168TIFF2025541705000043.tif248168TIFF2025541705000044.tif248168TIFF2025541705000045.tif131168

[0210] Cell type: HEP3B cell line cells Drug solvent: enzyme-free sterile water, Gibco DMEM (purchased from Thermo Fisher Scientific, catalog number: 10569010)

[0211] 2. Experimental Method The inhibitory effect of the samples on the mRNA expression of PCSK9 gene in HEP3B cell line was measured using qRT-PCR. Hep3B cells were provided by Wuxi Apptec Co., Ltd. (ATCC-tings-1618164).

[0212] 2.1 Cell culture Subcultured HEP3B cell line was collected. Cells in the logarithmic growth phase were cultured in 10% fetal bovine serum RPMI 1640 medium (supplemented with 10 μL / mL each of penicillin and streptomycin) in an incubator at 37°C and 5% CO2, with the medium changed once daily. Cells were subcultured by digesting with 0.25% trypsin and centrifuging at 1000 rpm for 5 minutes. The supernatant was discarded and new medium was added for subculture.

[0213] 2.2 Cell transfection Preparation of transfection mixture: Lipofectamine RNAiMAX and Opti-MEM were mixed in a ratio of 1.5:98.5, and then mixed using a vortex mixer. Preparation of transfection reagent: 60 μL of siRNA solution diluted with Opti-MEM was added to 60 μL of transfection mixture at a 1:1 (v / v) ratio, mixed with a vortex mixer, and left at room temperature for 15 min to obtain lipid nanoparticles (LNPs). 12.5 μL of LNPs were used for encapsulation efficiency measurements. Transfection reagent for blank control group: 60 μL of the prepared transfection mixture was added to 60 μL of Opti-MEM, mixed with a vortex mixer, and allowed to stand at room temperature for 15 minutes. The prepared transfection reagent was added to a 24-well cell culture plate (100 μL / well) so that the final siRNA concentration was 0.05 nM / well. The cell suspension (6 × 10 4After mixing using the cross method, the mixture was placed in an incubator at 37°C and 5% CO2 for 40 hours.

[0214] 2.3 PCSK9 mRNA measurement The steps were the same as those in "2.3 Measurement of PCSK9 mRNA" in Example 2. 2.4 Data Processing The PCSK9 mRNA expression rate (%) was calculated as follows. Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) x 100% Inhibition rate of PCSK9 gene expression = 1 - expression rate (%).

[0215] 2.5 EC50 experiment In this experiment, the siRNA concentration of each sequence in the EC50 experiment was 0.2 nM, followed by 4-fold dilutions to a total of eight concentration points (0.2 nM, 0.05 nM, 0.0125 nM, 3.13 pM, 0.78 pM, 0.2 pM, 0.05 pM, and 0.01 pM). The inhibition rate of each sequence at each concentration was measured and plotted to calculate the EC50 concentration of each sequence.

[0216] 3. Experimental Results After the experiment was repeated three times, the inhibition rates of each modified sequence against the PCSK9 gene in Hep3B cells are shown in Tables 14 to 25. The activity assay results showed that the 12 candidate sequences (unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83) had significant inhibitory effects on the PCSK9 gene after being modified with the modified templates DV25 to DV29 designed in the present disclosure, with the inhibition rates all exceeding 70%. Among them, the inhibition rates of P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 reached 89.3%, 89.3%, and 89.1%, respectively.

[0217] Sequences modified with the modification templates DV25 to 29 of the present disclosure exhibited significantly improved inhibition rates of PCSK9 gene expression compared to alternately modified sequences. For example, the sequence P92-si51-DV27, obtained by modifying base sequence 51 with template DV27, exhibited a 29.1% improved inhibition rate compared to the alternately modified sequence, and the sequence P92-si81-DV26, obtained by modifying base sequence 81 with template DV26, exhibited a 26.9% improved inhibition rate compared to the alternately modified sequence.

[0218] Furthermore, the activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when the base sequence 51 was modified with the template DV27 of the present disclosure, the inhibition rate was 22.8% higher than when the sequence was modified with the modified template DV31 of the present disclosure, and 21.3% higher than when the sequence was modified with the known Advanced ESC template DV21.

[0219] According to EC50 experiments, the sequences modified with the modified templates designed in the present disclosure had EC50 values ​​of 0.0001 nM to 0.005 nM. For example, the EC50 values ​​of P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. These base sequences were found to be able to effectively inhibit PCSK9 gene expression even at low concentrations.

[0220] (i) Inhibitory effect of template-modified base sequence on PCSK9 gene (1) Basic sequence 5

[0221] [Table 14]

[0222] I. DV25-29 Templates of this Disclosure When base sequence 5 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 80%, which was significantly higher than that when alternating 2'-methoxy and 2'-fluoro modifications were performed. For example, the inhibition rates of P92-si5-DV26 and P92-si5-DV27 obtained by modification with templates DV26 and DV27 were improved by 19.5% and 16.6%, respectively (Figure 7).

[0223] II. Other Modified Templates of the Disclosure After modifying base sequence 5 with templates DV30 and DV31, the inhibition rates of P92-si5-DV30 and P92-si5-DV31 against the PCSK9 gene were 65.3% and 62.4%, respectively, which were 0.1% and 3.0% lower than those of the corresponding alternately modified sequences, respectively. P92-si5-DV26 and P92-si5-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates, 19.6% and 16.7% higher than P92-si5-DV30 and 22.5% and 19.6% higher than P92-si5-DV31.

[0224] III. Advanced ESC templates DV21 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, 11, 16, and 17 of the sense strand) and DV22 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, and 11 of the sense strand) disclosed in the prior art P92-si5-DV21 and P92-si5-DV22, obtained by modifying base sequence 5 with templates DV21 and DV22, had inhibitory rates of 68.2% and 69.1%, respectively, against the PCSK9 gene, which were 2.8% and 3.7%, respectively, higher than the corresponding alternately modified sequences. P92-si5-DV26 and P92-si5-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates, 16.7% and 13.8% higher than P92-si5-DV21 and 15.8% and 12.9% higher than P92-si5-DV22.

[0225] summary: 1) When base sequence 5 was modified with modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified. For example, when base sequence 5 was modified with DV26, the inhibition rate was improved by 19.5% compared to when the sequence was alternately modified. 2) The activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when the sequence of base sequence 5 was modified with the modification templates DV26 and DV27 of the present disclosure, the inhibition rate increased by up to 22.5%, significantly higher than when the sequence was modified with the modification templates DV30 and DV31 of the present disclosure. The inhibition rate increased by up to 16.7%, significantly higher than when the sequence was modified with the known Advanced ESC templates DV21 and DV22. 3) siRNA sequences modified with different templates showed very large activity differences of up to 20%. It was found that it is unclear which template modification is required to ensure high activity for siRNA sequences.

[0226] (2) Base sequence 51 [Table 15]

[0227] I. DV25-29 Templates of this Disclosure After modification of base sequence 51 with the modification templates DV25-29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 75%, which was significantly improved compared to the case of alternating 2'-methoxy and 2'-fluoro modifications. For example, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 obtained by modification with templates DV26 and DV27 were improved by 27.7% and 29.1%, respectively.

[0228] II. Other Modified Templates of the Disclosure P92-si51-DV30 and P92-si51-DV31, obtained by modifying base sequence 51 with templates DV30 and DV31, had inhibitory rates of 63.5% and 62.8%, respectively, against the PCSK9 gene, which were only 7.0% and 6.3%, respectively, higher than the corresponding alternating modified sequences. P92-si51-DV26 and P92-si51-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates, 20.7% and 22.1% higher than P92-si51-DV30 and 21.4% and 22.8% higher than P92-si51-DV31.

[0229] III. Advanced ESC templates DV21 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, 11, 16, and 17 of the sense strand) and DV22 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, and 11 of the sense strand) disclosed in the prior art P92-si51-DV21 and P92-si51-DV22, obtained by modifying base sequence 51 with the known Advanced ESC templates DV21 and DV22, had inhibitory rates of 64.3% and 65.1%, respectively, against the PCSK9 gene, which were 7.8% and 8.6%, respectively, higher than the alternatively modified sequences. P92-si51-DV26 and P92-si51-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates, 19.9% ​​and 21.3% higher than P92-si51-DV21 and 19.1% and 20.5% higher than P92-si5-DV22.

[0230] summary: 1) When the base sequence 51 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified. For example, when the base sequence 51 was modified with DV27, the inhibition rate was improved by 29.1% compared to when the sequence was alternately modified. 2) The activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when the base sequence 51 was modified with the modification templates DV26 and DV27 of the present disclosure, the inhibition rate increased by up to 22.8%, significantly more than when the sequence was modified with the modification templates DV30 and DV31 of the present disclosure. Furthermore, the inhibition rate increased by up to 21.3%, significantly more than when the sequence was modified with the known Advanced ESC templates DV21 and DV22. 3) siRNA sequences modified with different templates showed very large activity differences of up to 20%. It was found that it is unclear which template modification is required to ensure high activity for siRNA sequences. (3) Base sequence 81

[0231] [Table 16]

[0232] I. DV25-29 Templates of this Disclosure When base sequence 81 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate against the PCSK9 gene exceeded 80%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-Si81-DV26 and P92-Si81-DV27, obtained by modifying the sequence with templates DV26 and DV27, were improved by 26.9% and 24.7%, respectively.

[0233] II. Other Modified Templates of the Disclosure P92-Si81-DV30 and P92-Si81-DV31, obtained by modifying base sequence 81 with templates DV30 and DV31, had inhibitory rates of 71.6% and 73.5%, respectively, against the PCSK9 gene, which were 9.2% and 11.1%, respectively, higher than the sequences obtained by the corresponding alternating modifications.

[0234] P92-Si81-DV26 and P92-Si81-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates of 17.7% and 15.5% compared to P92-Si81-DV30, and 15.8% and 13.6% compared to P92-Si81-DV31.

[0235] III. Advanced ESC templates DV21 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, 11, 16, and 17 of the sense strand) and DV22 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, and 11 of the sense strand) disclosed in the prior art P92-Si81-DV21 and P92-Si81-DV22, obtained by modifying base sequence 81 with templates DV21 and DV22, had inhibitory rates of 78.3% and 75.1%, respectively, against the PCSK9 gene, which were 15.9% and 12.7%, respectively, higher than those of the corresponding sequences obtained by alternating modification. P92-Si81-DV26 and P92-Si81-DV27, obtained by modifying the sequences with the modified templates DV26 and DV27 of the present disclosure, showed significantly improved inhibition rates of 11.0% and 8.8% compared to P92-Si81-DV21, and 14.2% and 12.0% compared to P92-Si81-DV22.

[0236] summary: 1) When the base sequence 81 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified. For example, when the base sequence 81 was modified with DV26, the inhibition rate was improved by 26.9% compared to when the sequence was alternately modified. 2) The activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when the base sequence 81 was modified with the modification templates DV26 and DV27 of the present disclosure, the inhibition rate increased by up to 17.7%, significantly more than when the sequence was modified with the modification templates DV30 and DV31 of the present disclosure. Furthermore, the inhibition rate increased by up to 14.2%, significantly more than when the sequence was modified with the known Advanced ESC templates DV21 and DV22. 3) siRNA sequences modified with different templates exhibited significantly different activities, with differences of up to 20%. It remains unclear which template modification is required to ensure high activity for siRNA sequences.

[0237] (4) Base sequence 82

[0238] [Table 17]

[0239] I. DV25-29 Templates of this Disclosure When base sequence 82 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 80%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-si82-DV27 and P92-si82-DV29 obtained by modification with templates DV27 and DV29 were improved by 19.6% and 19.4%, respectively.

[0240] II. Other Modified Templates of the Disclosure P92-si82-DV30 and P92-si82-DV31, obtained by modifying base sequence 82 with templates DV30 and DV31, had inhibitory rates of 70.2% and 69.7%, respectively, against the PCSK9 gene, corresponding to the corresponding alternately modified sequences. The modified templates P92-si82-DV27 and P92-si82-DV29 obtained by modifying the sequences of the modified templates DV27 and DV29 of the present disclosure showed significantly improved inhibition rates of 19.1% and 18.9% compared to P92-si82-DV30, and 19.6% and 19.4% compared to P92-si82-DV31.

[0241] III. Advanced ESC templates DV21 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, 11, 16, and 17 of the sense strand) and DV22 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, and 11 of the sense strand) disclosed in the prior art P92-si82-DV21 and P92-si82-DV22, obtained by modifying base sequence 82 with templates DV21 and DV22, had inhibitory rates of 76.5% and 75.4%, respectively, against the PCSK9 gene, which were 6.8% and 5.7%, respectively, higher than the alternately modified sequences. The modified templates P2-si82-DV27 and P92-si82-DV29 obtained by modifying the DV27 and DV29 sequences of the present disclosure showed significantly improved inhibition rates of 12.8% and 12.6% compared to P92-si82-DV21, and 13.9% and 13.7% compared to P92-si82-DV22.

[0242] summary: 1) When the base sequence 82 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified. For example, when the base sequence 82 was modified with DV27, the inhibition rate was improved by 19.6% compared to when the sequence was alternately modified. 2) The activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when base sequence 82 was modified with the modification templates DV27 and DV29 of the present disclosure, the inhibition rate increased by up to 19.6%, significantly more than when the sequence was modified with the modification templates DV30 and DV31 of the present disclosure. Furthermore, the inhibition rate increased by up to 13.9%, significantly more than when the sequence was modified with the known Advanced ESC templates DV21 and DV22. 3) siRNA sequences modified with different templates showed very large activity differences of up to 20%. It was found that it is unclear which template modification is required to ensure high activity for siRNA sequences.

[0243] (5) Base sequence 84

[0244] [Table 18]

[0245] I. DV25-29 Templates of this Disclosure When the base sequence was modified with the modification templates DV25-29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 85%, which was significantly higher than when the base sequence was modified with alternating 2'-methoxy and 2'-fluoro. For example, the inhibition rates of P92-Si84-DV28 and P92-Si84-DV29 obtained by modification with templates DV28 and DV29 were improved by 26.8% and 25.9%, respectively.

[0246] II. Other Modified Templates of the Disclosure P92-Si84-DV30 and P92-Si84-DV31, obtained by modifying base sequence 84 with templates DV30 and DV31, had inhibitory rates of 70.3% and 72.4%, respectively, against the PCSK9 gene, which were 8.7% and 10.8%, respectively, higher than the corresponding alternating modified sequences. The modified templates P2-Si84-DV28 and P92-Si84-DV29 obtained by modifying the DV28 and DV29 sequences of the present disclosure showed significantly improved inhibition rates of 18.1% and 17.2% higher than those of P92-Si84-DV30, and 16.0% and 15.1% higher than those of P92-Si84-DV31.

[0247] III. Advanced ESC templates DV21 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, 11, 16, and 17 of the sense strand) and DV22 (fluorinated sites: positions 2, 6, 14, and 16 of the antisense strand, and positions 7, 9, 10, and 11 of the sense strand) disclosed in the prior art P92-Si84-DV21 and P92-Si84-DV22, obtained by modifying base sequence 84 with templates DV21 and DV22, had inhibitory rates of 77.7% and 75.1%, respectively, against the PCSK9 gene, which were 16.1% and 13.5%, respectively, higher than the alternately modified sequences. The modified templates P2-si84-DV28 and P92-si84-DV29 obtained by modifying the DV28 and DV29 sequences of the present disclosure showed significantly improved inhibition rates of 10.7% and 9.8% compared to P92-si84-DV21, and 13.3% and 12.4% compared to P92-si84-DV22.

[0248] summary: 1) When the base sequence 84 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified. For example, when the base sequence 84 was modified with DV28, the inhibition rate was improved by 26.8% compared to when the sequence was alternately modified. 2) The activity of the same siRNA sequence was significantly different after modification with different modification templates. For example, when the base sequence 84 was modified with the modification templates DV28 and DV29 of the present disclosure, the inhibition rate increased by up to 18.1%, significantly more than when the sequence was modified with the modification templates DV30 and DV31 of the present disclosure. Furthermore, the inhibition rate increased by up to 13.3%, significantly more than when the sequence was modified with the known Advanced ESC templates DV21 and DV22. 3) siRNA sequences modified with different templates showed very large activity differences of up to 20%. It was found that it is unclear which template modification is required to ensure high activity for siRNA sequences.

[0249] (6) Basic sequence 3

[0250] [Table 19]

[0251] 1) When basic sequence 3 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 75%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-Si3-DV28 and P92-Si3-DV29 obtained by modification with templates DV28 and DV29 were improved by 24.0% and 24.7%, respectively. 2) It was found that when the sequence of basic sequence 3 was modified with modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0252] (7) Basic Sequence 8

[0253] [Table 20]

[0254] 1) When basic sequence 8 was modified with the modification templates DV25 to 29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 75%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-Si8-DV27 and P92-Si8-DV28 obtained by modification with templates DV27 and DV28 were improved by 19.7% and 20.2%, respectively. 2) It was found that when the sequence of basic sequence 8 was modified with modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0255] (8) Basic Sequence 9

[0256] [Table 21]

[0257] 1) When base sequence 9 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 70%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-Si9-DV27 and P92-Si9-DV28 obtained by modification with templates DV27 and DV28 were improved by 27.2% and 25.4%, respectively. 2) It was found that when the base sequence 9 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0258] (9) Base Sequence 21

[0259] [Table 22]

[0260] 1) When base sequence 21 was modified with the modification templates DV25 to 29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 70%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-si21-DV25 and P92-si21-DV26 obtained by modification with templates DV25 and DV26 were improved by 24.4% and 26.5%, respectively. 2) It was found that when the base sequence 21 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0261] (10) Base Sequence 31

[0262] [Table 23]

[0263] 1) When base sequence 31 was modified with the modification templates DV25 to DV29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 70%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-si31-DV25 and P92-si31-DV26 obtained by modification with templates DV25 and DV26 were improved by 22.9% and 22.6%, respectively. 2) It was found that when the base sequence 31 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0264] (11) Base sequence 73

[0265] [Table 24]

[0266] 1) When base sequence 73 was modified with the modification templates DV25-29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 70%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-si73-DV28 and P92-si73-DV29 obtained by modification with templates DV28 and DV29 were improved by 30.6% and 32.7%, respectively. 2) It was found that when the base sequence 73 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0267] (12) Base sequence 83

[0268] [Table 25]

[0269] 1) When base sequence 83 was modified with the modification templates DV25-29 designed in the present disclosure, the inhibition rate of the PCSK9 gene exceeded 75%, which was significantly higher than when alternating modifications with 2'-methoxy and 2'-fluoro were performed. For example, the inhibition rates of P92-Si83-DV27 and P92-Si83-DV29 obtained by modification with templates DV27 and DV29 were improved by 30.9% and 29.7%, respectively. 2) It was found that when the base sequence 83 was modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect on the PCSK9 gene was significantly improved compared to when the sequence was alternately modified.

[0270] summary: (1) The selected base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83 were modified with the modified templates DV25-29 designed in the present disclosure and showed significant inhibitory effects on PCSK9 gene expression, with inhibition rates of all of them exceeding 70%. Among them, the inhibition rates of P92-Si81-DV26, P92-Si82-DV27, and P92-Si81-DV29 reached 89.3%, 89.3%, and 89.1%, respectively. (2) The 12 sequences modified with the modification templates DV25 to 29 of the present disclosure exhibited significantly improved inhibition rates of PCSK9 gene expression compared to the alternately modified sequences. For example, P92-Si51-DV27, obtained by modifying base sequence 51 with template DV27, exhibited a 29.1% improved inhibition rate compared to the alternately modified sequence, and P92-Si81-DV26, obtained by modifying base sequence 81 with template DV26, exhibited a 26.9% improved inhibition rate compared to the alternately modified sequence. (3) When the same sequence was modified with the modification templates DV25 to 29 of the present disclosure, the inhibition rate of PCSK9 gene expression was significantly improved compared to when the same sequence was modified with the modification templates disclosed in the prior art. For example, when the base sequence 51 was modified with the modification template DV27 of the present disclosure, the inhibition rate was improved by 21.3% compared to when the sequence was modified with the known Advanced ESC template DV21. (4) When the same sequence was modified with the modification templates DV25 to 29 of the present disclosure, the inhibition rate of PCSK9 gene expression was significantly improved compared to when the same sequence was modified with other modification templates DV30 and DV31 of the present disclosure. For example, when the base sequence 51 was modified with the modification template DV27 of the present disclosure, the inhibition rate was improved by 22.8% compared to when the base sequence was modified with the modification template DV31 of the present disclosure. (5) Sequences modified with different templates showed very different activity effects. For example, when base sequence 5 was modified with DV26, the inhibition rate was 22.5% higher than when it was modified with DV31. When base sequence 51 was modified with DV27, the inhibition rate was 20.5% higher than when it was modified with DV22. It was found that it is unclear which modification template can be used to modify siRNA sequences to achieve high activity.

[0271] (ii) EC50 experiment A total of 12 template-modified sequences were selected, including P92-si5-DV26, P92-si51-DV25, P92-si81-DV27, P92-si82-DV27, P92-si84-DV26, P92-si3-DV28, P92-si8-DV29, P92-si9-DV28, P92-si21-DV25, P92-si31-DV27, P92-si73-DV28, and P92-si83-DV26. EC50 experiments were performed to determine the EC50 concentration of each sequence. The experimental results are shown in Table 26.

[0272] [Table 26]

[0273] Table 26 shows that the EC50 values ​​of these 12 sequences ranged from 0.0001 nM to 0.005 nM. Among them, the EC50 values ​​of P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. These results indicate that these sequences can effectively inhibit PCSK9 gene expression even at low concentrations.

[0274] Example 5: Comparison of the inhibitory effect of sequences disclosed in the prior art on the PCSK9 gene In this example, the inhibitory efficiency against the PCSK9 gene was compared between unmodified sequences disclosed in the prior art that are identical or similar to the base sequences 3, 5, 8, 9, 31, 81, 82, 83, and 84 disclosed herein, and the alternating modified sequences disclosed herein and sequences modified with modified templates DV25-29.

[0275] 1. Experimental Materials sample: (1) Sequences disclosed in the prior art

[0276] [Table 27]

[0277] Note: All of the sequences disclosed in the above patent applications were unmodified RNA sequences.

[0278] (2) In Example 2, siRNA sequences were used, such as P92-si5, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si31, and P92-si83, which were alternately modified with 2'-OMe and 2'-F and had their ends modified with thiol. (3) siRNA sequences modified with modification templates DV25 to 29 (details of the sequences are shown in Table 28) were used.

[0279] [Table 28] TIFF2025541705000061.tif243168TIFF2025541705000062.tif243168 TIFF2025541705000063.tif122168

[0280] Cell type: Hep3B cells (ATCC-tings-1618164) provided by Wuxi Apptec Co., Ltd.

[0281] Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061). Drug solvent: enzyme-free sterile water, Gibco DMEM.

[0282] 2. Experimental Method The inhibitory effect of the samples on the mRNA expression of the PCSK9 gene in HEP3B cell line was measured using qRT-PCR.

[0283] 2.1 Cell culture Subcultured HEP3B cell line was collected. Logarithmically growing cells were cultured in 10% fetal bovine serum RPMI 1640 medium (supplemented with 10 μL / mL each of penicillin and streptomycin) in a 37°C, 5% CO2 incubator, with the medium changed once daily. Cells were subcultured by digestion with 0.25% trypsin. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and new medium was added for subculture.

[0284] 2.2 Cell transfection Two microliters of 1 μg / μL siRNA was added to 18 μL of enzyme-free sterile water at a ratio of 1:0.06 (wt / wt) and mixed. 22 μL of the prepared LNP was added, mixed, and allowed to stand at room temperature for 15 minutes. 12.5 μL was used to measure encapsulation efficiency. The final transfection amount / well = (1 μg theoretical sample drop amount / siRNA purity) / drug loading concentration, and the final siRNA concentration was 0.05 nM. Negative control group: 22 μL of the prepared LNP was added to 20 μL of enzyme-free sterile water, mixed, and allowed to stand at room temperature for 15 minutes. After mixing using the cross method, the cells were cultured in an incubator at 37°C and 5% CO2 for 40 hours.

[0285] 2.3 PCSK9 mRNA measurement 1) RNA extraction The steps were the same as those in "1) RNA extraction" in "2.3 Measurement of PCSK9 mRNA" in Example 2. 2) Measurement of RNA concentration The steps were the same as those in "2) Measurement of RNA concentration" in "2.3 Measurement of PCSK9 mRNA" in Example 2. 3) Quantitative measurement of PCSK9 mRNA In a 15 mL centrifuge tube, other components except primers and template were added at 7.5 x 84 = 630 parts and marked as A. Marked 1.5 mL EP tubes. Add 77 μL of A and 420 ng of total RNA (7 parts / tube) and mix (marked as B). The upstream and downstream primers of the internal control gene GAPDH and the target gene PCSK9 were mixed (marked as C). 11 μL of B+1 μL of C was added to each well of the PCR plate, covered with sealing film, centrifuged at 3000 rpm for 1 minute, and transferred to the device. *This step was performed on ice to maintain cold conditions. The plate was placed in a qPCR machine and run according to the following program: Reverse transcription: 55°C, 15 minutes. Heat denaturation: 95°C, 30 seconds. Cycle reaction: 95°C, 10 seconds, 60°C, 35 seconds, 40 cycles. Melting curve: 95°C, 15 seconds. 60°C, 60 seconds. 95°C, 15 seconds. The run time was approximately 2 hours. The experimental results were analyzed and 2-ΔΔCt was calculated.

[0286] 2.4 Data Processing The PCSK9 mRNA expression rate (%) was calculated as follows. Expression rate = (PCSK9 mRNA expression level in the experimental group / PCSK9 mRNA expression level in the blank control group) x 100% Inhibition rate of PCSK9 gene expression = 1 - expression rate (%).

[0287] 3. Experimental Results Specific experimental results are shown in Tables 29 and 30. Experimental results show that the alternating modified sequences and sequences modified with specific modification templates of the present disclosure have significantly improved inhibitory effects on PCSK9 compared to the same or similar unmodified sequences disclosed in the prior art. For example, the same unmodified sequence si84 had an inhibition rate of 38.6%, but the inhibition rate after alternating modification was 61.6%, an inhibition rate 23.0% higher than that of the unmodified sequence si84. After modification with the modification template DV26 of the present disclosure, the inhibition rate reached 86.8%, an inhibition rate 48.2% higher than that of the unmodified sequence.

[0288] The sequence 31P disclosed in the prior art is different from the sequence si31 of the present disclosure in that only the two bases UG at the 3' end of the antisense strand are deleted, and the remaining bases are exactly the same. However, the unmodified sequence si31 of the present disclosure had an inhibition rate 10.4% higher than 31P, the inhibition rate of the alternately modified sequence was 59.5%, which was 21.2% higher than 31P, and the inhibition rate after modification with the modified template DV26 of the present disclosure reached 80.2%, which was 41.9% higher than 31P.

[0289] (1) The alternating modified sequences and sequences modified with specific modified templates of the present disclosure have significantly improved inhibition rates against the PCSK9 gene, up to 40%, compared to the identical unmodified sequences disclosed in the prior art. The alternating modified sequence of the present disclosure and the sequence modified with specific modified template have significantly improved inhibition rate against PCSK9 gene compared with the same unmodified sequence disclosed in the prior art.For example, the unmodified sequence si84 has an inhibition rate of 38.6%, but after alternating modification, the inhibition rate is 61.6%, which is 23.0% higher than the unmodified sequence si84, and after modification with the modified template DV26 of the present disclosure, the inhibition rate reaches 86.8%, which is 48.2% higher than the unmodified sequence.

[0290] [Table 29]

[0291] The unmodified sequences si81, si84, si3, si8, si9 and si83 disclosed in the prior art in Table 29 were identical to the sequences of the present disclosure, but when alternately modified and modified with the modified templates of the present disclosure, their inhibitory effects on PCSK9 were significantly improved. For example, the unmodified sequence si84 had an inhibition rate of 38.6%, while the alternatingly modified P92-si84 had an inhibition rate of 61.6%, which was 23.0% higher than the unmodified sequence si84. The modified P92-si84-DV25, P92-si84-DV26, P92-si84-DV27, P92-si84-DV28 and P92-si84-DV29 obtained by modification with the modified templates DV25 to 29 of the present disclosure had inhibition rates of 84.9%, 86.8%, 84.5%, 86.2% and 86.4%, respectively, all of which were more than 40% higher than the unmodified sequence si84.

[0292] (2) The unmodified sequences, alternately modified sequences, and sequences modified with specific modified templates disclosed herein have significantly improved inhibition rates against the PCSK9 gene, up to 40%, compared to unmodified sequences with only minor differences disclosed in the prior art. The unmodified sequences, alternately modified sequences, and sequences modified with specific modified templates of the present disclosure have significantly improved inhibition rates against the PCSK9 gene compared to the sequences disclosed in the prior art that have only slight differences. For example, the unmodified sequence 31P had an inhibition rate of 38.3%, while the similar unmodified sequence si31 of the present disclosure had an inhibition rate of 48.7%, which was 10.4% higher than 31P; the alternately modified sequence had an inhibition rate of 59.5%, which was 21.2% higher than 31P; and the sequence obtained by modification with the modified template DV26 of the present disclosure reached an inhibition rate of 80.2%, which was 41.9% higher than 31P.

[0293] [Table 30]

[0294] I. Sequence Comparison The sequences 5P, 31P, and 82P disclosed in the prior art in Table 30 were very close to the sequences si5, si31, and si82 of the present disclosure, with only minor differences. Specific comparisons are shown in the table below.

[0295] [Table 31]

[0296] From Table 31, it can be seen that sequence 5P disclosed in the prior art lacks only two bases, GG, at the 3' end of the antisense strand compared to sequence si31 of the present disclosure. Sequence 31P lacks only two bases, UG, at the 3' end of the antisense strand compared to sequence si31 of the present disclosure. Sequence 82P lacks only two bases, AA, at the 3' end of the antisense strand compared to sequence si82 of the present disclosure, but the remaining bases are exactly the same.

[0297] II. Activity comparison 1) The unmodified sequences of the present disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 of the present disclosure exhibits a 10.4% improved inhibition rate compared to the similarly structured 31P. The inhibition rate of the unmodified sequence si5 of the present disclosure against the PCSK9 gene was 50.1%, which was a significant improvement of 8.3% over the similar sequence 5P disclosed in the prior art (inhibition rate 41.8%). The inhibition rate of the unmodified sequence si82 of the present disclosure against the PCSK9 gene was 53.8%, which was significantly improved by 7.1% compared to the similar sequence 82P disclosed in the prior art (inhibition rate of 46.7%). The inhibition rate of the unmodified sequence si31 of the present disclosure against the PCSK9 gene was 48.7%, which was a significant improvement of 10.4% over the similar sequence 31P disclosed in the prior art (inhibition rate 38.3%).

[0298] 2) The alternating modified sequences of the present disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the alternating modified sequence P92-si5 of the present disclosure exhibited a 22.0% improved inhibition rate compared to sequence 5P. P92-si5, obtained by alternately modifying the basic sequence 5 of the present disclosure, had an inhibition rate of 63.8% against the PCSK9 gene, which was significantly improved by 22.0% compared to the unmodified sequence 5P disclosed in the prior art.

[0299] P92-si82, obtained by alternately modifying the basic sequence 82 of the present disclosure, had an inhibition rate of 68.3% against the PCSK9 gene, which was significantly improved by 21.6% compared to the unmodified sequence 82P disclosed in the prior art. P92-si31, obtained by alternately modifying the basic sequence 31 of the present disclosure, had an inhibition rate of 59.5% against the PCSK9 gene, which was significantly improved by 21.2% compared to the unmodified sequence 31P disclosed in the prior art.

[0300] 3) Sequences modified with the modified templates of the present disclosure exhibit significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the alternating modified sequence P92-si82-DV29 of the present disclosure exhibited a 42.8% improvement in inhibition over sequence 82P. The basic sequence 5 modified with the modified templates 25 to 29 of the present disclosure all had inhibition rates 30% or more higher than the unmodified sequence 5P disclosed in the prior art. For example, the inhibition rate of P92-si5-DV27 was significantly improved by 40.8%. The basic sequence 82 modified with the modification templates 25 to 29 of the present disclosure all had inhibition rates 30% or more higher than the unmodified sequence 82P disclosed in the prior art. For example, the inhibition rate of P92-si82-DV29 was significantly improved by 42.8%. The basic sequence 31 modified with the modified templates 25 to 29 of the present disclosure all had inhibition rates 30% or more higher than the unmodified sequence 31P disclosed in the prior art. For example, the inhibition rate of P92-si31-DV26 was significantly improved by 41.9%.

[0301] This shows that the unmodified sequences, alternately modified sequences, and template-modified sequences disclosed herein have significantly improved inhibitory activity against PCSK9 compared to similar unmodified sequences disclosed in the prior art, with the inhibition rate increasing by up to 40%.

[0302] summary: (1) The alternating modified sequences and sequences modified with specific modified templates of the present disclosure have significantly improved inhibitory effects on the PCSK9 gene compared to the same sequences disclosed in the prior art. For example, the unmodified sequence si84 had an inhibitory rate of 38.6%, while the inhibitory rate after alternating modification was 61.6%, a 23.0% improvement over the unmodified sequence si84. The inhibitory rate after modification with the modified template DV26 of the present disclosure reached 86.8%, a 48.2% improvement over the unmodified sequence. (2) The unmodified sequences, alternately modified sequences, and sequences modified with specific modification templates of the present disclosure exhibit significantly improved inhibitory effects on the PCSK9 gene compared to similar sequences disclosed in the prior art. For example, the unmodified sequence si31 of the present disclosure exhibited a 10.4% improved inhibition rate compared to the similar sequence 31P disclosed in the prior art. The alternately modified sequence P92-si5 of the sequence si5 of the present disclosure exhibited a 22.0% improved inhibition rate compared to the unmodified sequence 5P disclosed in the prior art, which is similar to sequence si5. The sequence P92-si82-DV29, obtained by modifying the sequence si82 of the present disclosure with the modification template DV29, exhibited a 42.8% improved inhibition rate compared to the unmodified sequence 82P disclosed in the prior art, which is similar to sequence si82.

[0303] Example 6: Inhibitory effects of sequences with specific anti-off-target designs on PCSK9 and off-target genes In practical applications of siRNA, the expression of non-target mRNAs that are only partially complementary to the guide strand (antisense strand) is often suppressed. Research by Alnylam has shown that the hepatotoxicity of N-acetylgalactosamine (GalNAc)-conjugated siRNA is primarily due to off-target effects that result in gene suppression of the wrong target via a recognition mechanism similar to that of microRNA (miRNA).

[0304] To address this issue, Alnylam has developed a new generation 5 template design that significantly reduces off-target effects and hepatotoxicity by disrupting the seed region of the siRNA antisense strand using glycol nucleic acid (GNA) modifications at position 7 of the antisense strand. This modification significantly reduces off-target effects and hepatotoxicity by disrupting the siRNA's seed region-mediated binding to undesigned targets.

[0305] Natural 5'-end phosphorylation or simple direct 5'-end phosphorylation can be dephosphorylated in cells. Directly 5'-end phosphorylated oligonucleotides are 90% dephosphorylated after circulating in the blood for 2 hours and completely disappear after 24 hours. The 5'-end phosphorylation design (5'-E-VP) uses E-vinylphosphonate ester instead of bridging oxygen, resulting in the most effective phosphorylation and higher stability.

[0306] Based on the above, in order to reduce the off-target effect of the sequence and to investigate the influence of 5'-end phosphorylation on the off-target effect, in this example, the base sequences 5, 51, 81, and 84 modified with DV25-29 were used, and the 5'-end of the antisense strand was modified with 5'-E-VP and the GNA anti-off-target design at position 7 was added.

[0307] Furthermore, to investigate off-target effects, a comparison was made between the alternating 2'-methoxy and 2'-fluoro modified sequences P92-si3, P92-si8, P92-si21, P92-si31, P92-si73 and P92-si83 and the above sequences P92-si3+, P92-si8+, P92-si21+, P92-si31+, P92-si73+ and P92-si83+ containing the GNA anti-off-target design at position 7 of the antisense strand.

[0308] Experimental results showed that at a concentration of 10 nM, the off-target modified sequences all had significant inhibitory effects on the target gene PCSK9, but the inhibitory effects on the off-target gene were significantly reduced. This indicates that the anti-off-target design does not affect the inhibitory effects of the alternating modified sequence and template modified sequence of the present disclosure on the PCSK9 gene, but can significantly inhibit the off-target effects.

[0309] 1. Experimental Materials 1) Sample: Alternately modified sequences: Alternately modified sequences P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73 and P92-si83. Sequences with alternate modifications and anti-off-target designs: P92-si3+, P92-si8+, P92-si9+, P92-si21+, P92-si31+, P92-si73+ and P92-si83+ (Table 32).

[0310] Table 33 shows the sequences obtained by performing template modification on base sequences 5, 84, 81, and 51, the sequences obtained by performing template modification and 5'-E-VP modification, the sequences obtained by performing template modification and anti-off-target design, and the sequences obtained by performing template modification, 5'-E-VP modification, and anti-off-target design.

[0311] 2) Sequence synthesis: I. Alternately modified sequences and template modified sequences The synthesis method was as described in Example 1. II. siRNA sequences containing 5'-E-VP The siRNA sequence was synthesized with reference to Example 1. When synthesizing the base at the 5' end of the antisense strand (the last base), a monomer having a phosphonic acid group at the 5' end, such as vinyl-(E)-phosphonate ester-A-OMe phosphoramidite monomer (Formula 9) or vinyl-(E)-phosphonate ester-U-OMe phosphoramidite monomer (Formula 10), was used. Their structural formulas were as follows: [ka]

[0312] III. siRNA sequences with anti-off-target design The siRNA sequence was synthesized with reference to Example 1. When synthesizing the seventh base from the 5' end of the antisense strand, the sequence was synthesized using a GNA monomer. The structural formula of the GNA monomer was as follows: [ka]

[0313] [Table 32]

[0314] The above sequences all had alternating 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) modifications. (1) The sense strand had 2'-F at all odd-numbered positions and 2'-OMe at all even-numbered positions. (2) The antisense strand had 2'-OMe at all odd-numbered positions and 2'-F at all even-numbered positions. (3) There were two thiol modifications at the 5' end of the sense strand and at the 5' and 3' ends of the antisense strand. (4) The seventh base (5'-3' direction) of the antisense strand was modified with GNA.

[0315] [Table 33] TIFF2025541705000071.tif242168TIFF2025541705000072.tif55168

[0316] Cell type: Hep3B cells (ATCC-tings-1618164) provided by Wuxi Apptec Co., Ltd.

[0317] Hep3B cells were cultured in EMEM medium (ATCC-30-2003) containing 10% fetal bovine serum (FBS, ExCell Bio-FSP500), 1% penicillin-streptomycin (HyClone-SV30010), 1% non-essential amino acid solution (Gibco-11140-050), and 1% GlutaMAX supplement (Gibco-35050-061). Drug solvent: enzyme-free sterile water, Gibco DMEM.

[0318] 2. Experimental Method 1) Compound dilution For off-target experiments, samples were diluted to a concentration of 10 nM and detected in triplicate wells. Digestion and counting of Hep3B cells: Hep3B cells at 80% confluency were washed with Dulbecco's phosphate-buffered saline (DPBS) and digested with 0.05% trypsin for 3–10 min. After harvesting, the cells were counted using a Countstar Rigel S2. The cell density was determined to be 2 × 10 5 / mL.

[0319] 2) Preparation of transfection reagent RNAiMAX transfection reagent was prepared. The appropriate amount was prepared in a 15mL centrifuge tube at a ratio of 3:97 RNAiMAX:Opti-MEM. The mixture was vortexed for 15 seconds and incubated at room temperature for 15 minutes. 40μL of RNAiMAX Opti-MEM was added to each well in the appropriate position, and 40μL of diluted compound at the appropriate concentration was added to the corresponding well of the dilution plate. The mixture was then mixed and incubated for 15 minutes.

[0320] 3) Cell plating Hep3B cells (2 × 10 4 100 cells / well) were plated in a 96-well cell culture plate, and siRNA compounds were mixed with RNAiMAX Opti-MEM transfection reagent and added to the cells in each well, along with a control group containing RNAiMAX Opti-MEM but no siRNA compounds.

[0321] Anti-off-target experiments: The compound and RNAiMAX Opti-MEM mixture was removed from the dilution plate and added to a 96-well cell culture plate (20 μL / well). Cells were then added to the 96-well plate at 100 μL / well for a final volume of 120 μL per well. After plating, the plates were cultured in a 5% CO2, 37°C incubator for 24 hours.

[0322] 4) RNA extraction and reverse transcription 24 hours after transfection, the medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy® 96 Kit (QIAGEN-74182) according to the kit's instructions. cDNA was then synthesized using the FastKing RT Kit (with gDNase) (TIANGEN-KR116-02) according to the kit's instructions.

[0323] 5) RT-qPCR Target cDNA was measured by qPCR, with GAPDH cDNA used as an internal standard in parallel. Eight μL of the prepared qPCR reaction mixture and 2 μL of sample cDNA were added to a 384-well plate. The TaqMan qPCR reaction consisted of heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds and 60°C for 1 minute, for a total of 40 cycles. The SYBR qPCR reaction consisted of heating at 50°C for 2 minutes, 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds and 60°C for 1 minute, for a total of 40 cycles. A final melting curve was obtained by heating at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0324] 6) Data analysis The RNA expression level of the target gene in the sample was calculated using the ΔΔCt relative quantification method based on the Ct value of each sample. The relative expression level of the target gene was expressed as 2 − ΔΔCT. It was calculated as follows: ΔCT = average Ct value of target gene - average Ct value of internal control gene ΔΔCT = ΔCT (treated group) - ΔCT (RNAiMAX control group) Relative expression level of target gene mRNA = 2-ΔΔCT Inhibition rate = (1 - relative expression level of sample / average expression level of RNAiMAX control group) x 100%

[0325] 3. Experimental Results (1) All of the different modified sequences had significant inhibitory effects on the PCSK9 gene. For example, the inhibitory rate of the alternately modified sequence P92-si3 reached 75.6%, and the inhibitory rate of the sequence D84-DV27P, which used template modification and 5'-E-VP modification, was as high as 94.4%. The inhibition rates of all samples against the PCSK9 gene are shown in Tables 34 to 36. All of the alternatively modified sequences had significant inhibitory effects on PCSK9 at a concentration of 10 nM. For example, the inhibitory rate of the alternatively modified sequence P92-si3 reached 75.6%.

[0326] I. The alternating modified sequences had a significant inhibitory effect on the PCSK9 gene, with the inhibition rate exceeding 60%, among which P92-si3 reached 75.6%.

[0327] [Table 34]

[0328] From the above table, at a concentration of 10 nM, the alternating modified sequences P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si51, P92-si73 and P92-si83 all had significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 60%, among which the inhibition rate of P92-si3 reached a maximum of 75.6%.

[0329] II. Template-modified sequences using anti-off-target design and / or 5'-E-VP modification had a significant inhibitory effect on the PCSK9 gene. For example, the inhibition rate of the sequence D84-DV27P using template modification and 5'-E-VP modification was as high as 94.4%.

[0330] [Table 35]

[0331] 1) The base sequences 5, 51, 81, and 84 modified with the modified templates of the present disclosure exhibited significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 90%. Furthermore, the inhibitory effects were even more potent than those of the alternately modified sequences. For example, P92-si51, obtained by alternately modifying base sequence 51, had an inhibition rate of 70.1% (see Table 34), while D51-DV26, obtained by modifying template DV26, had an inhibition rate of 90.7%, a significant improvement of 20.6% over the alternately modified sequences (Figure 8).

[0332] 2) Template modification followed by anti-off-target design demonstrated significant inhibitory activity against PCSK9 gene expression. For example, D84-DV26+, obtained by modifying base sequence 84 with template DV26 followed by anti-off-target design, showed a high inhibitory rate of 90.1%.

[0333] 3) Using template modification followed by 5'-E-VP modification can improve the inhibitory effect on the PCSK9 gene. For example, D84-DV27, obtained by modifying base sequence 84 with template DV27, had an inhibitory rate of 91.5%. Further 5'-E-VP modification resulted in D84-DV27P, which had an inhibitory rate of 94.4%, an improvement of 2.9%.

[0334] 4) By using template modification followed by anti-off-target design and 5'-E-VP modification, the inhibitory activity against the PCSK9 gene can reach over 90%. For example, D84-DV27+P, which was obtained by modifying base sequence 84 with template DV27 followed by anti-off-target and 5'-E-VP modification, achieved an inhibitory rate of 90.4%.

[0335] From the above, when the sequence is modified with the modification templates DV25 to 29 of the present disclosure, the inhibitory effect can be further enhanced compared to when the sequence is alternately modified. Furthermore, it was found that when the sequence is modified with the modification templates DV25 to 29 of the present disclosure, followed by anti-off-target design and / or 5'-E-VP modification, a significant inhibitory effect on PCSK9 gene expression is exhibited, with an inhibition rate of 90% or more.

[0336] (2) When anti-off-target design was further performed in addition to any one or more of the various modified forms of the present disclosure, it had a significant anti-off-target effect on off-target genes and reduced the inhibitory effect on off-target genes. The experimental results revealed the following: 1) When anti-off-target design is performed on the alternatively modified sequence or template modified sequence of the present disclosure, the inhibitory effect on off-target genes can be reduced, i.e., they have a significant anti-off-target effect.

[0337] 2) When both anti-off-target design and 5'-E-VP modification were used in sequences with template modifications, the inhibition rate against off-target genes was significantly reduced, i.e., it was found to have a significant anti-off-target effect.

[0338] I. When the anti-off-target design is used in the alternatively modified sequence of the present disclosure, it has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0339] [Table 36]

[0340] Table 36 shows that after the anti-off-target design is used in the alternating modified sequence of the present disclosure, the inhibition rate of off-target genes is reduced, indicating an anti-off-target effect. For example, the alternating modified sequence P92-Si3+ using the anti-off-target design has a 20.0% reduction in the inhibition rate of the off-target gene AARSD1 and a 20.5% reduction in the inhibition rate of the off-target gene ACAP2 compared to the sequence P92-Si3 without the anti-off-target design, showing a significant reduction.

[0341] II. When an anti-off-target design is used on a sequence modified with the modified template of the present disclosure, it has a significant anti-off-target effect, reducing the inhibition rate of off-target genes by up to 73.6%.

[0342] [Table 37]

[0343] 1) Use of off-target modifications only (indicated by a "+" in the SEQ ID NO:) Anti-off-target design using template modifications significantly reduced the inhibitory effect on off-target genes, for example, the inhibitory rate of D84-DV26+ against the MXD1 gene was reduced by 39.9%, that of D81-DV25+ against the PCYOX1 gene was reduced by 54.1%, that of D51-DV26+ against the NEPRO gene was reduced by 25.9%, and that of D5-DV25+ against the DTWD1 gene was reduced by 10.9%.

[0344] Modification of base sequences 5, 51, 81 and 84 with the modification templates designed in this disclosure, followed by further anti-off-target design, has been shown to have a significant anti-off-target effect.

[0345] 2) Anti-off-target design combined with 5'-E-VP modification (indicated by "+P" in SEQ ID NO.) Sequences with both template and 5'-E-VP modifications exhibited significant anti-off-target effects, with significantly reduced inhibition of off-target genes. For example, D84-DV26+P significantly reduced the inhibition of off-target gene MXD1 by 73.6%, D81-DV27+P significantly reduced the inhibition of off-target gene PCYOX1 by 31.1%, and D51-DV26+P significantly reduced the inhibition of off-target gene KIF1B by 41.6%.

[0346] Modification of base sequences 5, 51, 81 and 84 with the modification templates designed in this disclosure, followed by further anti-off-target design and 5'-E-VP modification, has been shown to have a significant anti-off-target effect.

[0347] summary: 1. All of the different modified sequences had significant inhibitory effects on the PCSK9 gene. For example, the inhibitory rate of the alternately modified P92-si3 reached 75.6%, and the inhibitory rate of the sequence D84-DV27P, which used template modification and 5'-E-VP modification, was as high as 94.4%. (1) The alternating modified sequences had a significant inhibitory effect on the PCSK9 gene, with the inhibition rate exceeding 60%, among which P92-si3 reached 75.6%. (2) Anti-off-target design and / or 5'-E-VP modification of template-modified sequences significantly inhibited the PCSK9 gene. For example, the sequence D84-DV27P, which used template modification and 5'-E-VP modification, had a high inhibition rate of 94.4%.

[0348] 2. When any one or more of the various modified forms of the present disclosure were further subjected to anti-off-target design, it had a significant anti-off-target effect on off-target genes and reduced the inhibitory effect on off-target genes. (1) The use of an anti-off-target design in the alternatively modified sequence of the present disclosure has a significant anti-off-target effect, reducing the inhibition rate of off-target genes by 20%. (2) When an anti-off-target design is used on a sequence modified with the modification template of the present disclosure, it has a significant anti-off-target effect, reducing the inhibition rate of off-target genes by up to 73.6%.

[0349] Example 7: Effect of sequences modified with the modified template of the present disclosure on PCSK9, low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in mouse serum In this example, several sequences including base sequences 5, 51, 81, 82, and 84 were selected as examples. These sequences were subjected to modifications such as template modification alone, a combination of template modification and anti-off-target design, a combination of template modification and 5'-E-VP modification, or a combination of template modification, 5'-E-VP modification, and anti-off-target design. Using transgenic mice expressing the human PCSK9 gene, the inhibitory effects of each of the above sequences on serum PCSK9, low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) were measured by ELISA at different time points.

[0350] 1. Experimental Materials Test drug: The sequences in Table 38 included sequences with template modification alone, sequences with template modification combined with anti-off-target design, sequences with template modification combined with 5'-E-VP modification, and sequences with template modification combined with 5'-E-VP modification combined with anti-off-target design. The GalNAc ligand G4, G5, G6, or G7 was bound to the 3' end of the sense strand of these sequences. [ka]

[0351] The methods for conjugating the oligonucleotides to the ligands G4, G5, G6, and G7 were the same as those for preparing conjugates 4, 5, 6, and 7 in Example 3 of Patent CN116854754A. Specifically, YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were conjugated to the oligonucleotides. The synthesis methods for YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 were the same as those in Example 1 of the present application.

[0352] The oligonucleotide and the ligand formed a conjugate as shown below. [ka]

[0353] The specific sequence of each sequence is shown in Table 38. G4, G5, G6, and G7 in the SEQ ID NOs indicate that the sequence was bound to the GalNAc ligand G4, G5, G6, or G7.

[0354] [Table 38] TIFF2025541705000080.tif243168TIFF2025541705000081.tif106168

[0355] Preparation of test drug Drug solvent: PBS buffer Preparation conditions: sterile environment Labeling method: A label was affixed to the prepared dosage formulation, and the theme number, name, concentration, quantity, preparation date, preparer, and storage conditions were written on the outer container. Storage conditions: Samples were prepared immediately before use, and remaining samples were stored at -80°C.

[0356] Experimental Animal Information: Species / strain: hPCSK9 transgenic mouse Grade: SPF Gender: Male Quantity: 214 Age: 4 - 7 weeks old Weight: 18 - 20 g Source: Jiangsu Jicui Yakang Biotechnology Co., Ltd. (GemPharmatech co.Itd) Manufacturing License Number: SCXK (Jiangsu) 2018 - 0008

[0357] Institutional Animal Care and Use Committee (IACUC): The experimental animals were bred by Youji (Tianjin) Pharmaceutical Technology Co., Ltd. (License Number for Use: SYXK (Tianjin Binjiang) 2019 - 0002) after being received. This project was reviewed by the Experimental Animal Ethics Committee of Youji (Tianjin) Pharmaceutical Technology Co., Ltd., and the experimental process was strictly carried out in accordance with the requirements of IACUC to ensure animal welfare.

[0358] Breeding and Management: Breeding Conditions: The experimental animals were bred by Youji (Tianjin) Pharmaceutical Technology Co., Ltd. (License Number for Use: SYXK (Tianjin Binjiang) 2019 - 0002) after being received. They were bred in a breeding cage with dimensions of length × width × height = 29.0 cm × 18.5 cm × 13.0 cm. The temperature range was 20°C - 26°C, the humidity range was 40% to 70%, the ventilation rate was 15 times / hour or more, and the artificial lighting was set to 12 hours of light and 12 hours of darkness.

[0359] The breeding environmental conditions conformed to the national standard GB14925 - 2010 of the People's Republic of China and were managed by a packaged air - conditioning unit. The animals were allowed to eat and drink freely. The drinking water bottles and the drinking water in the bottles were replaced at least twice a week. After use, the drinking water bottles were sterilized by a pulsating vacuum sterilizer and reused.

[0360] The breeding cages and bedding of the animals were replaced at least once a week. All breeding cages and bedding of the animals were sterilized by a pulsating vacuum sterilizer and used in a barrier environment. The breeding cages of the animals were washed, disinfected, and wiped at least once a week. The observation room for animal breeding was washed and disinfected daily, including flat shelves, floors, tables, etc. <00​ As the disinfectant solutions used in the barrier environment, there are 6.67% benzalkonium bromide solution, 0.5% 84 disinfectant solution, 75% disinfectant solution, and 0.08% BESTAQUAM-S. The four types of disinfectants were used in sequence and must not be mixed.

[0362] Feed for experimental animals: SPF rat and mouse maintenance feed (manufactured by Spf (Beijing) Biotechnology Co., Ltd., and the animal feed production license number SCXK (Beijing) 2019-0010 issued by the Beijing Municipal Science and Technology Commission). Feed inspection: Each batch of feed has a quality certificate, and our company conducted a microbial inspection once every quarter. The feed supplier submitted the latest third-party feed inspection report every six months. The feed nutrient component inspection referred to the National Standard of the People's Republic of China GB14924.3-2010, and the contaminant index inspection referred to the National Standard of the People's Republic of China GB14924.2-2001.

[0363] Drinking water for experimental animals: Sterilized water prepared by a filtration system and directly filled into drinking water bottles. Drinking water inspection: Our company conducted a microbial experiment once every quarter and sent water to a third-party inspection agency once a year for water quality inspection. The drinking water inspection referred to the National Standard of the People's Republic of China GB5749-2006.

[0364] Dressings for animals: Corn cob (manufactured by Spf (Beijing) Biotechnology Co., Ltd., and the animal dressing production license number SCXK (Beijing) 2019-0004 issued by the Beijing Municipal Science and Technology Commission). Dressing inspection: Our company conducted a microbial experiment once every quarter. The dressing supplier submitted at least one third-party dressing inspection report every six months. The dressing inspection referred to the National Standard of the People's Republic of China GB14924.2-2001.

[0365] 2. Experimental methods <00018!>Dosage setting and grouping Definition of experimental day: The day when the solvent or test drug was administered to the animals was defined as day 0. Grouping and administration: After adaptive breeding, the experimental animals were randomly divided into a negative control group and a test drug group according to the serum PSCK9 protein content, with 5 animals per group. A single subcutaneous administration of 6 mg / kg was administered in a volume of 1 mL / kg at a concentration of 6 mg / mL. The day of administration was designated as day 0.

[0366] Animals were individually identified by ear tags. Cages were identified by hanging cage cards. A laboratory sign was hung on the laboratory door. Details of grouping are shown in the table below.

[0367] [Table 39]

[0368] indicator measurement (1) General Observations The animals were observed once a day from one week before administration until the end of the experiment. Observation contents: Animals were observed near the cage for death or moribund status, mental state, behavior and activity, fecal properties, and food and drinking water supply status. Experimental animals: All animals in the negative control and test drug groups.

[0369] (2) Expression of PCSK9 protein in serum Measurement times: 3 days before administration on day-3 (day-3), 1 week (1w) after administration on day-7, 2 weeks (2w) after administration on day-14, 3 weeks (3w) after administration on day-21, 4 weeks (4w) after administration on day-28, and 5 weeks (5w) after administration on day-35. PCSK9 protein levels were measured using an ELISA kit. Serum samples were kept freezing and thawing-free. Experimental animals: All animals in the negative control and test drug groups.

[0370] (3) Lipid measurement Measurement times: Approximately 200 μL of blood was collected from the medial canthus of the eye 3 days before administration on day 3 (day 3), 1 week after administration on day 7 (1 week), 2 weeks after administration on day 14 (2 weeks), 3 weeks after administration on day 21 (3 weeks), 4 weeks after administration on day 28 (4 weeks), and 5 weeks after administration on day 35 (5 weeks). The whole blood samples were temporarily stored in an icebox before centrifugation. After centrifugation at 2-8°C and approximately 3000 g for 10 minutes, the samples were divided into two tubes (approximately 60 μL in one tube and the remaining serum in the other tube) and stored at -70 to -86°C for lipid analysis.

[0371] (4) Data processing and statistical analysis The experimental data are expressed as mean ± standard deviation (Mean ± SD) and analyzed using GraphPad Prism 8.3 software. The LSD test was used to check for homogeneity of variance, and the Dunnett T3 test was used to check for unequal variance. P < 0.05 was considered statistically significant.

[0372] 3. Experimental Results Specific experimental results are shown in Tables 40 to 42. Tables 40-42 show that these sequences can sustainably and significantly inhibit serum PCSK9 protein and significantly reduce serum low-density lipoprotein cholesterol (LDL-C) and serum total cholesterol (TC) levels. For example, in the D81-DV25G7 group, the inhibition rates of serum PCSK9 protein reached 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively. In the D82-DV27G5 group, serum LDL-C levels decreased by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively. In the D82-DV29G7 group, serum TC levels decreased by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[0373] (1) The sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84 with different modifications, had significant inhibitory effects on serum PCSK9 protein expression. For example, in the D81-DV25G7 group, the inhibition rates reached 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively.

[0374] [Table 40]

[0375] Table 40 shows that the conjugates formed by conjugating each sequence with GalNAc compounds had significant inhibitory effects on serum PCSK9 protein expression. For example, the inhibitory rates of D81-DV25G7 reached 83.1%, 82.2%, and 70.3% at 7, 14, and 28, respectively (Figure 9).

[0376] Base sequences designed in this disclosure (e.g., base sequences 5, 51, 81, 82, and 84) have been shown to be capable of being efficiently delivered to animal livers and significantly inhibiting PCSK9 gene expression when conjugated with GalNAc compounds using the template modifications of this disclosure, or simultaneously using 5'-E-VP modifications and / or anti-off-target designs.

[0377] (2) The sequences of the present disclosure, such as base sequences 5, 51, 81, 82, and 84 with different modifications, can significantly reduce serum low-density lipoprotein cholesterol (LDL-C) levels. For example, in the D82-DV27PG5 group, LDL-C levels were reduced by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively.

[0378] [Table 41]

[0379] Table 41 shows that serum LDL-C levels were significantly reduced in each experimental group. For example, in the D82-DV27PG5 group, LDL-C levels were reduced by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively (Figure 10). The base sequences designed in this disclosure (e.g., 5, 51, 81, 82, and 84) have been shown to be efficiently delivered to animal livers and significantly reduce serum low-density lipoprotein cholesterol (LDL-C) levels when conjugated with GalNAc compounds using the template modifications of this disclosure, or simultaneously using 5'-E-VP modifications and / or anti-off-target designs.

[0380] (3) The sequences of the present disclosure, such as base sequences 5, 51, 81, 82, and 84 with different modifications, can significantly reduce serum total cholesterol (TC) levels. For example, D82-DV29PG7 reduced TC levels by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[0381] [Table 42]

[0382] Table 42 shows that serum total cholesterol (TC) levels were significantly reduced in each experimental group. For example, in the D82-DV29PG7 group, TC levels were reduced by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively (FIG. 11). The base sequences designed in this disclosure (e.g., base sequences 5, 51, 81, 82, and 84) have been shown to be efficiently delivered to animal livers and significantly reduce serum total cholesterol (TC) levels when conjugated with GalNAc compounds using the template modifications of this disclosure, or simultaneously using 5'-E-VP modifications and / or anti-off-target designs.

[0383] summary: The base sequences designed in this disclosure (e.g., base sequences 5, 51, 81, 82, and 84) can be efficiently delivered to animal livers when conjugated with GalNAc compounds using the template modifications of this disclosure, or simultaneously using 5'-E-VP modifications and / or anti-off-target designs, and can sustainably and significantly inhibit serum PCSK9 protein expression and reduce serum low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels.

[0384] For example, in the D81-DV25G7 group, the inhibition rates of serum PCSK9 protein expression reached 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively. In the D82-DV27PG5 group, serum low-density lipoprotein cholesterol (LDL-C) levels decreased by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively. In the D82-DV29PG7 group, serum total cholesterol (TC) levels decreased by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[0385] Conclusion: In this disclosure, we designed a series of siRNAs based on the PCSK9 mRNA sequence, and modified these sequences using a set of specific modification templates through alternating modifications, and further modified some of the sequences with anti-off-target design and 5'-E-VP modifications. The results are as follows: (1) Unmodified sequences containing the base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83 all had significant inhibitory effects on PCSK9, with inhibition rates exceeding 60%.

[0386] (2) The inhibition rate of the sequence with multiple modifications reached up to 90% or more. Furthermore, when conjugated with GalNAc compounds, the modified sequence could be efficiently delivered to the animal liver, where it significantly inhibited PCSK9 gene expression, significantly reduced serum low-density lipoprotein cholesterol (LDL-C) levels, and significantly reduced serum total cholesterol (TC) levels.

[0387] Details are as follows:

[0388] 1. The alternatively modified sequences of the present disclosure had a significant inhibitory effect on the PCSK9 gene. (1) Of the 84 sequences designed, 12 sequences, including P92-si5, P92-si51, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73, and P92-si83, exhibited significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among these, P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exhibited inhibition rates exceeding 70%. (2) The 26 sequences had inhibitory rates of 30% to 50% against the PCSK9 gene. For example, the inhibitory rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively. (3) Forty-six sequences showed an inhibitory rate of 30% or less against the PCSK9 gene. For example, the inhibitory rates of P92-si1 and P92-si20 were 9.2% and 6.0%, respectively. (4) siRNAs with similar sequences showed very different activities. For example, P92-si5 showed a significantly improved inhibitory rate of 61.8% compared with P92-si4.

[0389] 2. The unmodified sequence of the present disclosure had a significant inhibitory effect on the PCSK9 gene. (1) Twelve unmodified sequences, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, had significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%. Among them, the inhibition rates of si5, si81, si82, si3, and si8 exceeded 60%. (2) Other unmodified sequences showed less than 40% inhibition of the PCSK9 gene, e.g., si52 and si74 showed only 0.5% and 2.2%, respectively. (3) siRNAs with similar sequences showed very different activities. For example, base sequence 5 showed a significantly improved inhibitory rate of 51.2% compared with base sequence 4. (4) Alternative modification of different sequences had varying effects on activity. For example, alternating modification of base sequence 5 resulted in a significant increase in inhibition (12.4%) compared to the unmodified sequence. Alternating modification of base sequences 4, 22, and 52 resulted in little change in inhibition compared to the unmodified sequence.

[0390] 3. The sequences modified with the modified templates of the present disclosure had a significant inhibitory effect on the PCSK9 gene. (1) The base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83 modified with the modified templates DV25-29 designed in this disclosure exhibited significant inhibitory effects on PCSK9 gene expression, with inhibition rates of 70% or higher. Among them, the inhibition rates of P92-si-DV26, P92-si82-DV27, and P92-si82-DV29 reached 89.3%, 89.3%, and 89.1%, respectively. (2) The 12 sequences modified with the modification templates DV25 to 29 of the present disclosure exhibited significantly improved inhibition rates of PCSK9 gene expression compared to the alternately modified sequences. For example, P92-si51-DV27, obtained by modifying the base sequence 51 with template DV27, exhibited a 29.1% improved inhibition rate compared to the alternately modified sequence. P92-si81-DV26, obtained by modifying the base sequence 81 with template DV26, exhibited a 26.9% improved inhibition rate compared to the alternately modified sequence. (3) When the same sequence was modified with the modification templates DV25 to 29 of the present disclosure, the inhibition rate of PCSK9 gene expression was significantly improved compared to when the same sequence was modified with the modification templates disclosed in the prior art. For example, when the base sequence 51 was modified with the modification template DV27 of the present disclosure, the inhibition rate was improved by 21.3% compared to when the sequence was modified with the known Advanced ESC template DV21. (4) When the same sequence was modified with the modification templates DV25 to DV29 of the present disclosure, the inhibition rate of PCSK9 gene expression was significantly improved compared to when the same sequence was modified with other modification templates DV30 and DV31 of the present disclosure. For example, when the base sequence 51 was modified with the modification template DV27 of the present disclosure, the inhibition rate was improved by 22.8% compared to when the sequence was modified with the modification template DV31 of the present disclosure. (5) The EC50 values ​​of the above 12 sequences ranged from 0.0001 nM to 0.005 nM, and the EC50 values ​​of P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. These sequences have been shown to be able to effectively inhibit PCSK9 gene expression even at low concentrations.

[0391] 4. The sequences modified with the alternating modifications and modified templates of the present disclosure exhibited significantly improved inhibitory activity against PCSK9 compared to unmodified sequences disclosed in the prior art that were completely identical or had only minor differences. (1) The alternating modified sequences and sequences modified with specific modified templates disclosed herein have significantly improved inhibition rates against the PCSK9 gene, up to 40%, compared to the completely identical unmodified sequences disclosed in the prior art. (2) The unmodified sequences, alternately modified sequences, and sequences modified with specific modified templates disclosed herein have significantly improved inhibition rates against the PCSK9 gene, up to 40%, compared to unmodified sequences with only minor differences disclosed in the prior art.

[0392] I. The unmodified sequences of the present disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 of the present disclosure exhibits a 10.4% improved inhibition rate compared to the similarly structured 31P. II. The alternating modified sequences of the present disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the alternating modified sequence P92-si5 of the present disclosure has a 22.0% improved inhibitory rate compared to sequence 5P. III. Sequences modified with the modified templates of the present disclosure exhibit significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the alternating modified sequence P92-si82-DV29 of the present disclosure exhibited a 42.8% improvement in inhibition compared to the sequence 82P.

[0393] 5. When specific anti-off-target designs were performed on the alternating modified sequence and template modified sequence of the present disclosure, they had a significant inhibitory effect on the PCSK9 gene and significantly reduced the inhibitory effect on off-target genes. (1) All of the different modified sequences had significant inhibitory effects on the PCSK9 gene. For example, the inhibitory rate of the alternately modified P92-si3 reached 75.6%, and the inhibitory rate of the sequence D84-DV27P, which used template modification and 5'-E-VP modification, was as high as 94.4%.

[0394] I. The alternating modified sequences had a significant inhibitory effect on the PCSK9 gene, with an inhibition rate of over 60%, among which P92-si3 reached 75.6%. II. Anti-off-target design and / or 5'-E-VP modification of template-modified sequences had a significant inhibitory effect on the PCSK9 gene. For example, the sequence D84-DV27P, which was template-modified and 5'-E-VP-modified, had a high inhibition rate of 94.4%.

[0395] (2) When anti-off-target design was further performed in addition to any one or more of the various modified forms of the present disclosure, it had a significant anti-off-target effect on off-target genes and reduced the inhibitory effect on off-target genes.

[0396] I. When the anti-off-target design is used in the alternatively modified sequence of the present disclosure, it has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%. II. When an anti-off-target design is used on a sequence modified with the modified template of the present disclosure, it has a significant anti-off-target effect, reducing the inhibition rate of off-target genes by up to 73.6%.

[0397] 6. The sequence modified with the modified template of the present disclosure significantly inhibited PCSK9 expression in mouse serum and significantly reduced low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels. (1) Different modifications of the base sequences 5, 51, 81, 82, and 84 of the present disclosure had significant inhibitory effects on serum PCSK9 protein expression. For example, in the D81-DV25G7 group, the inhibition rates reached 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively. (2) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly reduced serum low-density lipoprotein cholesterol (LDL-C) levels. For example, in the D82-DV27PG5 group, LDL-C levels were reduced by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively. (3) Different modifications of the base sequences 5, 51, 81, 82, and 84 of the present disclosure could significantly reduce serum total cholesterol (TC) levels. For example, in the D82-DV29PG7 group, TC levels were reduced by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

Claims

1. A double-stranded RNAi agent comprising any one selected from the following double-stranded oligonucleotides in which the sense strand and the antisense strand match: (1) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 1 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand has the sequence shown in SEQ ID NO. 13 or a fragment thereof, or a modified version of said sequence or said fragment; (2) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 2 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand has the sequence shown in SEQ ID NO. 14 or a fragment thereof, or a modified version of said sequence or said fragment; (3) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 3 or a fragment thereof, or a modified sequence of said sequence or said fragment, and the antisense strand has the sequence shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence of said sequence or said fragment; (4) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 4 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (5) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (6) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 18 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (7) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 19 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (8) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (9) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 21 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (10) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 22 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (11) A double-stranded oligonucleotide in which the sense strand has the sequence shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof, and the antisense strand has the sequence shown in SEQ ID NO. 23 or a fragment thereof, or a modified sequence of said sequence or a fragment thereof; (12) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 12 or a fragment thereof, or a modified version of said sequence or said fragment, and the antisense strand has the sequence shown in SEQ ID NO. 24 or a fragment thereof, or a modified version of said sequence or said fragment.

2. 2. The double-stranded RNAi agent of claim 1, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides.

3. The double-stranded RNAi agent according to claim 1 or 2, which is an RNAi agent used to inhibit PCSK9 gene expression.

4. 3. The double-stranded RNAi agent of claim 1, wherein the sense strand differs from the sequence of any one of SEQ ID NOs. 1 to 12 by 1 to 3 nucleotides.

5. 3. The double-stranded RNAi agent of claim 1, wherein the antisense strand differs from the sequence of any one of SEQ ID NOs. 13 to 24 by 1 to 3 nucleotides.

6. At least one modified nucleotide is a deoxynucleotide, a 3'-terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy ...

3. The double-stranded RNAi agent of claim 1, wherein the nucleotide is selected from the group consisting of a nucleotide, an abasic nucleotide, a 2'-amino group-modified nucleotide, a 2'-O-allyl group-modified nucleotide, a 2'-C-alkyl group-modified nucleotide, a 2'-hydroxy group-modified nucleotide, a 2'-methoxyethyl group-modified nucleotide, a 2'-O-alkyl group-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl group-modified nucleotide, a thiophosphate group-containing nucleotide, a methyl phosphate group-containing nucleotide, a 5'-phosphate ester-containing nucleotide, and a 5'-phosphate ester analog-containing nucleotide.

7. 3. The double-stranded RNAi agent of claim 1 or 2, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

8. 3. The double-stranded RNAi agent of claim 1 or 2, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

9. The double-stranded RNAi agent of claim 1 or 2, wherein the double-stranded region has a length of 15 to 30 nucleotide pairs.

10. The double-stranded RNAi agent of claim 1 or 2, wherein the double-stranded region has a length of 17 to 25 nucleotide pairs.

11. The double-stranded RNAi agent of claim 1 or 2, wherein the double-stranded region has a length of 19 to 23 nucleotide pairs.

12. 3. The double-stranded RNAi agent of claim 1 or 2, wherein the double-stranded region has a length of 21 nucleotide pairs.

13. The double-stranded RNAi agent of claim 1 or 2, wherein each strand has 15 to 30 nucleotides.

14. The double-stranded RNAi agent of claim 1 or 2, wherein each strand has 19 to 25 nucleotides.

15. 3. The double-stranded RNAi agent of claim 1 or 2, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

16. The double-stranded RNAi agent according to claim 1 or 2, wherein the modified form of all nucleotides in the sense strand and the antisense strand is a chemical modification of the 2'-position of the ribose of the nucleotide.

17. The double-stranded RNAi agent according to claim 1 or 2, wherein the chemical modification at the 2'-position of the ribose of the nucleotide is any one or a combination of two or more selected from a 2'-methoxy group, a 2'-methoxyethyl group, a 2'-fluoro group, a 2'-benzyloxy group, a 2'-methylcarbonylamino group, and a 2'-pyridylmethoxy group.

18. The double-stranded RNAi agent according to claim 1 or 2, wherein the chemical modification at the 2'-position of the ribose of each nucleotide is selected from a combination of a 2'-methoxy group and a 2'-fluoro group.

19. The double-stranded RNAi agent according to claim 1 or 2, wherein the chemical modification at the 2'-position of the ribose of each nucleotide is selected from an alternating combination of a 2'-methoxy group and a 2'-fluoro group.

20. The double-stranded RNAi agent according to claim 1 or 2, wherein the chemical modification of the 2'-position of ribose in each nucleotide is 2'-fluoro modified at all odd-numbered positions in the sense strand, 2'-methoxy modified at all even-numbered positions in the sense strand, 2'-methoxy modified at all odd-numbered positions in the antisense strand, and 2'-fluoro modified at all even-numbered positions in the antisense strand.

21. The double-stranded RNAi agent according to claim 1 or 2, wherein the nucleotide monomers are linked together via a 3',5'-phosphodiester bond.

22. The double-stranded RNAi agent according to claim 1 or 2, wherein the nucleotide monomers are linked together via a thiolated 3',5'-phosphodiester bond.

23. The antisense strand is modified with any one of the following modifications A, B, and C, and the sense strand is modified with the following modification a or b, (In the above table, 2'-OMe represents a 2'-methoxy group, 2'-F represents a 2'-fluoro group, and PS represents a thiophosphate skeleton.) If modification A is used on the antisense strand, modification form a is used on the sense strand, When modification B is used in the antisense strand, modification form a is used in the sense strand, When modification C is used in the antisense strand, modification form a is used in the sense strand, If modification B is used on the antisense strand, modification form b is used on the sense strand, The double-stranded RNAi agent according to claim 1 or 2, further comprising a modification in which when modification C is used in the antisense strand, modification form b is used in the sense strand.

24. The double-stranded RNAi agent according to claim 1 or 2, wherein positions 2 to 8 from the 5' end of the antisense strand are modified with a group selected from UNA, GNA, and DNA, and the structures of UNA and GNA are as follows: 【Chemistry 1】 wherein the base is selected from adenine, guanine, cytosine, thymine, and uracil.

25. The double-stranded RNAi agent of claim 1 or 2, wherein the phosphorylation of the 5'-position carbon atom in the glycoside of the 5'-terminal nucleotide of the modified antisense strand may include, but is not limited to, the following 5'-position phosphorylation groups: a 5'-vinylphosphonate group (5'-E-VP), a 5'-methylphosphonate group (5'-MP), a 5'-C-methylphosphate group, a 5'-thiophosphate group (5'-PS), and a 5'-phosphate group (5'-P). 【Chemistry 2】 (wherein R is hydrogen, a hydroxyl group, an amine group, C １－４ Alkyl group, aryl group, C １～４ Alkoxy group, C １～４ alkylcarbonylamino group or halogen, The base is selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

26. 3. The double-stranded RNAi agent of claim 1 or 2, wherein the 3',5'-phosphodiester bonds linking the nucleotide monomers at the ends of the sequence are modified with thiol groups to form chirally pure 3',5'-phosphorothiodiester bonds, the 5'-ends of the sense strand and antisense strand contain 1 to 3 thiol groups, and the 3'-end of the antisense strand contains 1 to 3 thiol groups.

27. A conjugate for reducing PCSK9 expression, comprising the double-stranded RNAi agent of any one of claims 1 to 26 and a ligand conjugated thereto.

28. 28. The conjugate of claim 27, wherein the ligand is conjugated to the 3' or 5' end of the sense strand of the oligonucleotide.

29. 29. The conjugate of claim 27 or 28, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

30. 29. The conjugate of claim 27 or 28, wherein the ligand is represented by the following structural formula: 【Transformation 3】 wherein X is hydrogen or a hydroxyl protecting group including acetyl, benzoyl, and isobutyryl, or H; Y is an amine protecting group selected from the group consisting of formyl, acetyl, propionyl, n-butyryl, and isobutyryl, or H; n is an integer from 0 to 20; and q, r, and s are each independently an integer from 1 to 7.

31. 31. The conjugate of claim 30, wherein the ligand is represented by the following structural formula: 【Chemistry 4】

32. 29. The conjugate of claim 27 or 28, wherein the ligand is represented by the following structural formula: 【Transformation 5】 wherein X is oxygen, nitrogen, or sulfur; Y is an alkyl group or an aryl group; R １ is oxygen or sulfur, R ２ is a hydrogen atom, an amino group, or C １～４ Alkyl group, aryl group, C １～４ is an alkoxy group or a halogen; A is -(CH ２ ) ａ -, -(CH ２ CH ２ O) ｂ -, -((CH ２ ) ｃ NHCO) ｄ - or -((CH ２ ) ｃ CONH) ｄ - (wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5), B is -(CH ２ ) ｅ - (wherein e is an integer from 0 to 7), L is —CONH— or —NHCO—, X １ is -(CH ２ ) ｆ - or - (CH ２ CH ２ O) ｆ CH ２ - (wherein f is an integer from 1 to 5), X ２ is -(CH ２ ) ｇ - (wherein g is an integer from 1 to 6), Y １ is 0 or 1, Y ２ is 0, 1 or 2, Y ３ is 1, 2 or 3, m is an integer from 0 to 4, n is an integer from 0 to 4.

33. 33. The conjugate of claim 32, wherein the ligand is represented by the following: G4, G5, G6 or G7. 【Transformation 6】

34. 29. The conjugate of claim 27 or 28, having the structure shown below: 【Transformation 7】

35. 29. The conjugate of claim 27 or 28, wherein the double-stranded RNAi agent comprises any one selected from the following double-stranded oligonucleotides having matched sense and antisense strands and conjugated to ligands G4, G5, G6, or G7: (1) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 337 and the antisense strand has the sequence shown in SEQ ID NO. 427; (2) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 337 and the antisense strand has the sequence shown in SEQ ID NO. 428; (3) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 342 and the antisense strand has the sequence shown in SEQ ID NO. 381; (4) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 342 and the antisense strand has the sequence shown in SEQ ID NO. 430; (5) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 342 and the antisense strand has the sequence shown in SEQ ID NO. 431; (6) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 347 and the antisense strand has the sequence shown in SEQ ID NO. 384; (7) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 347 and the antisense strand has the sequence shown in SEQ ID NO. 437; (8) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 347 and the antisense strand has the sequence shown in SEQ ID NO. 438; (9) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 348 and the antisense strand has the sequence shown in SEQ ID NO. 385; (10) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 352 and the antisense strand has the sequence shown in SEQ ID NO. 448; (11) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO. 390; (12) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO. 448; (14) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO. 393; (15) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO. 446; (16) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 357 and the antisense strand has the sequence shown in SEQ ID NO.

447.

36. 29. The conjugate of claim 27 or 28, wherein the double-stranded RNAi agent comprises any one selected from the following double-stranded oligonucleotides having matched sense and antisense strands and conjugated to ligands G4, G5, G6, or G7: (1) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 352 and the antisense strand has the sequence shown in SEQ ID NO. 448; (2) a double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO. 390; (3) A double-stranded oligonucleotide, wherein the sense strand has the sequence shown in SEQ ID NO. 353 and the antisense strand has the sequence shown in SEQ ID NO.

448.

37. 29. The conjugate of claim 27 or 28, wherein the double-stranded RNAi agent comprises a double-stranded oligonucleotide comprising a sense strand set forth in SEQ ID NO. 353 and an antisense strand set forth in SEQ ID NO. 448, and conjugated to ligand G5.

38. 38. A pharmaceutical composition comprising the double-stranded RNAi agent of any one of claims 1 to 26 or the conjugate of any one of claims 27 to 37, and a pharmaceutically acceptable carrier.

39. 39. Use of the double-stranded RNAi agent of any one of claims 1 to 26, the conjugate of any one of claims 27 to 37, or the pharmaceutical composition of claim 38 in the manufacture of a medicament for the treatment of a PCSK9-related disease.

40. 40. The use of claim 39, wherein the PCSK9-related disease is selected from hypercholesterolemia, atherosclerosis, dyslipidemia, cardiovascular disease or cerebrovascular disease.

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