Sirna for targeted regulation of PCSK9 gene expression, and use thereof

Abstract

The invention relates to a siRNA for targeted regulation of PCSK9 gene expression and a use thereof. Experimental results show that some oligonucleotide sequences subject to alternate modification and specific template modification can significantly inhibit PCSK9 expression, and can be used for developing a drug for treating PCSK9-related diseases.

Description

siRNA targeting and regulating PCSK9 gene expression and its application

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on November 6, 2023, with application number 202311463375.7 and invention name “siRNA targeting and regulating PCSK9 gene expression and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to the field of nucleic acid modification technology, and in particular to a small interfering ribonucleic acid (siRNA) modified by multiple chemical methods and its application in the preparation of drugs for diseases related to PCSK9 gene expression. Background Art

[0003] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers.

[0004] Oligonucleotides are short DNA or RNA molecules or oligomers that readily bind in a sequence-specific manner to their respective complementary oligonucleotides, DNA, or RNA, forming duplexes or, less commonly, higher-order hybrids. This fundamental property has led to their widespread application in genetic testing, research, and medicine. In nature, oligonucleotides are often small RNA molecules that play a role in regulating gene expression or intermediates derived from the degradation of larger nucleic acid molecules.

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

[0006] Classic RNAi molecules are composed of a signature 19+2 nucleotide polymer structure (a double helix consisting of a 21-nucleotide RNA molecule and 19 corresponding nucleobases, with a 2-nucleotide 3' overhang). One strand of the siRNA (the guide or antisense strand) is complementary to the target gene's mRNA transcript, while the other strand is designated the passenger strand (or sense strand). The siRNA (antisense strand) guides the Argonaute protein (AGO2) to complement the target transcript and becomes part of the RNA-guided silencing complex (RISC). The perfect complementarity between the siRNA (antisense strand) and the target leads to cleavage of the target transcript at positions 10-11 opposite the guide (antisense) strand, catalyzed by the AGO2 protein.

[0007] siRNA possesses inherent advantages over small molecule and antibody drugs. SiRNA performs its function by performing Watson–Crick base pairing with mRNA, whereas small molecule and monoclonal antibody drugs must recognize the complex spatial structure of specific proteins. Consequently, many diseases are resistant to small molecule and monoclonal antibody treatments because their target molecules are highly active and cannot recognize molecular structures with affinity and binding specificity. The mechanism of action of siRNA drugs enables them to regulate the expression of target proteins at the genetic level, offering greater targeting specificity than small molecule or antibody drugs. Its mechanism, based on the principle of complementary base pairing, also broadens the therapeutic scope of siRNA, simplifies design, and shortens the development cycle.

[0008] Oligonucleotides can sequence-specifically bind to complementary RNA strands, and upon hybridization, they can induce RNase H to cleave target RNA. The nucleotides in natural oligonucleotides are linked by phosphodiester bonds. Under physiological conditions, they are particularly sensitive to nucleases. Therefore, natural, unmodified, and unstructured oligonucleotide drugs are easily and rapidly degraded by nucleases in vivo, resulting in low activity and poor drugability. Chemical modification of oligonucleotide structure is an effective way to enhance their activity, improving their stability to nucleases and affinity for RNA, and better promoting cellular endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0009] Based on the basic structure of oligonucleotides, bases, sugar rings, phosphate backbones and ends, chemical modifications can be performed in four parts:

[0010] 1) Base modifications: These are mainly categorized 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-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propyneuridine, and dihydrouridine.

[0011] 2) Sugar ring modification: This is mainly divided into sugar ring modification and replacement. Sugar ring modification includes 2'-modification, 4'-modification, 5'-modification, isomeric modification, and combinations of these modifications. The most common 2'-modifications of siRNA are 2'-OMe (2'-methoxy) and 2'-F (2'-fluoro) modifications. Compared to natural siRNA, siRNA modified with both 2'-OMe and 2'-F has a higher Tm value, stronger serum stability, and better activity.

[0012] 3) Modification of the phosphate backbone: The main modification methods include: modification with phosphorothioate; modification with methyl phosphate, selenophosphate, methylborylphosphate, dithiophosphate, and replacement of the bridging oxygen atom in the phosphodiester bond connection region with a sulfur atom; replacement of the entire phosphate group between nucleosides with a group that does not contain a phosphorus atom, such as replacing the P atom with a C, S, and N atom to form a guanidine group, S-methylthiourea, etc.

[0013] 4) Terminal modification: covalent conjugation of specific groups at the 5' end and / or 3' end of the sense strand and phosphorylation modification of the 5' end of the antisense strand.

[0014] All oligonucleotide drugs that have been marketed have been chemically modified. Since the first nucleic acid drug, Fomivirsen (Vitravene), was approved for marketing in 1998, the chemical modification technology of nucleic acid drugs has been continuously upgraded. To date, 18 nucleic acid drugs have been approved for marketing worldwide:

[0015] Table 1 Nucleic acid drugs approved for marketing worldwide

[0016] The treatment strategy for hypercholesterolemia primarily focuses on lowering atherogenic low-density lipoprotein cholesterol (LDL-C) and raising potentially cardioprotective high-density lipoprotein cholesterol (HDL-C). Clinical studies involving nearly 170,000 subjects have shown that for every 1.0 mmol / L reduction in LDL-C, the average annual incidence of major cardiovascular disease events decreases by approximately 20%. The 2019 edition of the "Guidelines for the Diagnosis and Treatment of Dyslipidemia" recommends LDL-C-lowering medications such as statins, cholesterol absorption inhibitors, robutecol, and PCSK9 inhibitors, with statins being the preferred treatment. The 2018 edition of the "Chinese Expert Consensus on Screening, Diagnosis, and Treatment of Familial Hypercholesterolemia" recommends statins and combination therapy with ezetimibe and PCSK9 inhibitors.

[0017] PCSK9 is a serine protease encoded by the PCSK9 gene and produced primarily in the liver. PCSK9 binds to the LDL receptor (LDL-R) on the surface of hepatocytes, degrading LDL-R. Because LDL-R binds to LDL-C in plasma and absorbs it into the liver (where the lipids are processed and excreted through bile), LDL-R degradation leads to elevated plasma LDL-C levels. PCSK9 inhibitors reduce PCSK9 expression, increasing LDL-R levels on the surface of liver cells and thereby reducing plasma LDL-C levels, ultimately lowering blood lipids.

[0018] In order to effectively treat hypercholesterolemia and dyslipidemia, there is a need in the art to further develop drugs that regulate PCSK9 gene expression.

[0019] Summary of the Invention

[0020] The present invention designs a series of unique siRNA sequences by targeting the PCSK9 mRNA sequence, and performs alternating modification and specific template modification on the sequences.

[0021] Typically, siRNA uses 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) to modify the monomer. However, even if only the above two monomer modifications are considered, for siRNA, the sense and antisense strands have a total of 44 bases, that is, there are 2 44 Furthermore, the number of possible modification schemes is even greater when different terminal thiolation configurations are added.

[0022] The activity of the same siRNA sequence can vary significantly depending on the modification method used. Even different siRNAs can have significantly different activities using the same modification method. While there are some principles for siRNA modification design, previous studies have shown that it is difficult to accurately predict activity based on the modification method, indicating that there is no definitive relationship between modification method and activity. Therefore, it is extremely difficult to select a highly active modification from the countless possible combinations.

[0023] The present invention screens out some alternatively modified and specially modified sequences that have a significant inhibitory effect on PCSK9 gene expression by chemically modifying the designed siRNA sequences.

[0024] In one aspect, the present disclosure provides a double-stranded RNAi agent for reducing the expression of PCSK9, comprising any one oligonucleotide duplex selected from the following sense and antisense strand pairs.

[0025] (1) The sense strand has a sequence as shown in SEQ ID NO. 1 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof;

[0026] (2) the sense strand has a sequence as shown in SEQ ID NO. 2 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof;

[0027] (3) the sense strand has a sequence as shown in SEQ ID NO. 3 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence thereof;

[0028] (4) the sense strand has a sequence as shown in SEQ ID NO. 4 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence thereof;

[0029] (5) the sense strand has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified sequence thereof;

[0030] (6) the sense strand has a sequence as shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 18 or a fragment thereof, or a modified sequence thereof;

[0031] (7) the sense strand has a sequence as shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 19 or a fragment thereof, or a modified sequence thereof;

[0032] (8) The sense strand has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified sequence thereof;

[0033] (9) the sense strand has a sequence as shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 21 or a fragment thereof, or a modified sequence thereof;

[0034] (10) The sense strand has a sequence as shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 22 or a fragment thereof, or a modified sequence thereof;

[0035] (11) the sense strand has a sequence as shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 23 or a fragment thereof, or a modified sequence thereof;

[0036] (12) The sense strand has a sequence as shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 24 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0037] In another aspect, the present disclosure also provides a conjugate for reducing the expression of PCSK9, comprising the double-stranded RNAi agent and a ligand conjugated thereto.

[0038] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the double-stranded RNAi agent or conjugate, and a pharmaceutically acceptable carrier.

[0039] In another aspect, the present disclosure also provides use of the aforementioned double-stranded RNAi agent, conjugate, or composition in the preparation of a drug for a disease associated with PCSK9.

[0040] In another aspect, the present disclosure also provides methods for treating or preventing diseases and conditions that can be modulated by downregulating PCSK9 gene expression.

[0041] The beneficial effects achieved by the present disclosure are at least as follows:

[0042] (1) Unmodified sequences, including basic sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, all have significant inhibitory effects on PCSK9, with the highest inhibition rate exceeding 60%.

[0043] (2) Using multiple modified sequences, the inhibition rate can reach up to 90%. Moreover, the modified sequence is conjugated with a GalNAc compound and can be efficiently delivered to the animal liver, significantly inhibiting PCSK9 gene expression, significantly reducing serum low-density lipoprotein cholesterol (LDL-C) levels, and significantly reducing serum total cholesterol (TC) levels.

[0044] (3) The sequences modified by alternating modification and modification template disclosed in the present invention have significantly improved PCSK9 inhibitory activity, up to 40%, compared with the unmodified sequences disclosed in the prior art that are completely identical or have very little difference.

[0045] (4) The present disclosure found that, regardless of whether the siRNA sequence was unmodified or alternating, the activity of similar siRNAs varied significantly. For example, the unmodified base sequence 5 exhibited a 51.2% higher inhibition rate than the base sequence 4, while the alternating modified sequence P92-si5 exhibited a 61.8% higher inhibition rate than P92-si4, representing significant improvements.

[0046] (5) The present disclosure also found that the effects on activity of different sequences after alternating modification were not consistent. Some showed significant increases in inhibition rate, such as base sequence 5, where the alternating modification increased the inhibition rate by 12.4% compared to the unmodified sequence. Others showed less significant increases, such as base sequences 4, 22, and 52, where the inhibition rates of the alternating modification sequences and the unmodified sequence remained essentially unchanged. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, rather than limiting the present disclosure.

[0048] FIG1 shows the alternatively modified sequences that have a significant inhibitory effect on the PCSK9 gene, and the inhibition rates of these sequences are all higher than 50%.

[0049] FIG2 shows the alternating modified sequences with an inhibition rate of 30% to 50% on the PCSK9 gene.

[0050] FIG3 shows the alternative modified sequences with an inhibition rate of less than 30% on the PCSK9 gene.

[0051] FIG4 shows the alternative modified sequences with an inhibition rate of less than 30% on the PCSK9 gene.

[0052] FIG5 shows the unmodified sequences that have a significant inhibitory effect on the PCSK9 gene, and the inhibition rates of these sequences are all higher than 50%.

[0053] FIG6 shows that the unmodified sequence has an inhibition rate of less than 40% on the PCSK9 gene.

[0054] FIG7 shows the inhibition rate of the PCSK9 gene after basic sequence 5 is modified with different templates.

[0055] FIG8 shows the inhibition rate of the PCSK9 gene by basic sequences 5, 51, 81 and 84 using template modification, anti-off-target and / or 5'-E-VP modification sequences.

[0056] FIG9 shows the inhibition rate of PCSK9 protein in animal serum after basic sequences 5, 51, 81, 82 and 84 were modified with different schemes and conjugated with GalNAc compounds.

[0057] FIG10 shows the effects of different modification schemes of basic sequences 5, 51, 81, 82 and 84 on the reduction of low-density lipoprotein cholesterol (LDL-C) in animal serum after conjugation with GalNAc compounds.

[0058] FIG11 shows that after basic sequences 5, 51, 81, 82 and 84 are modified with different schemes and conjugated with GalNAc compounds, the total cholesterol (TC) level in animal serum is reduced. DETAILED DESCRIPTION

[0059] In order to make this disclosure more easily understood, some terms are first defined. In addition, it should be noted that whenever a value or a range of values ​​for a parameter is listed, it is intended to indicate that values ​​and ranges intermediate to these cited values ​​are also intended to be part of this disclosure.

[0060] As used herein, the articles "a" and "an" refer to one or 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 more than one element, such as a plurality of elements.

[0061] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to."

[0062] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0063] As used herein, the term "approximately" or "approximately" as applied to one or more target values ​​refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or in addition apparent from context, the term "approximately" or "approximately" refers to a value falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) or less (unless such numerals will exceed 100% of possible values).

[0064] As used herein, "PCSK9" refers to the proprotein convertase subtilisin Kexin9 gene or protein.

[0065] "G," "C," "A," and "U" each generally represent a nucleotide comprising guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to a modified nucleotide (as further described below) or an alternative replacement moiety. The skilled artisan is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement moiety). For example, without limitation, a nucleotide comprising inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine may be substituted in the nucleotide sequences of the present disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such substituted moieties are embodiments of the present disclosure.

[0066] The terms "RNAi agent," "iRNA," "iRNA agent," and "RNA interference agent" are used interchangeably herein to refer to RNA agents, as those terms are defined herein, and 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 modulates, e.g., inhibits, the expression of PCSK9 in cells, such as cells in 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).

[0067] The term "small interfering RNA" or "siRNA" refers to a small interfering RNA RNAi molecule. It is a type of double-stranded RNA molecule, also known in the art as short interfering RNA or silencing RNA. siRNAs typically comprise a sense strand (also known as a passenger strand) and an antisense strand (also known as a leader strand), wherein each strand is 17 to 30 nucleotides in length, typically 19 to 25 nucleosides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to the target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region. The siRNA strands may form blunt-ended duplexes, or preferably, the 3' ends of the sense and antisense strands may form 3' overhangs, such as 1, 2, or 3 nucleosides, similar to the products produced by Dicer, which may form RISC substrates in vivo. Effective extended forms of Dicer substrates have been described in US 8,349,809 and US 8,513,207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have 2 nt 3' overhangs. Thus, the duplex region can be, for example, 17 to 25 nucleotides in length, such as 21 to 23 nucleotides in length.

[0068] The term "antisense strand" refers to a strand of an RNAi (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not fully complementary to the target sequence, mispairing can occur in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0069] The term "sense strand," as used herein, 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).

[0070] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0071] As used herein, the phrase "inhibiting the expression of PCSK9" includes inhibiting the expression of any PCSK9 gene (such as, for example, a mouse PCSK9 gene, a rat PCSK9 gene, a monkey PCSK9 gene, or a human PCSK9 gene) as well as variants (e.g., naturally occurring variants) or mutants of the PCSK9 gene. Thus, the PCSK9 gene can be a wild-type PCSK9 gene, a mutant PCSK9 gene, or a transgenic PCSK9 gene in the context of a genetically manipulated cell, cell group, or organism.

[0072] "Inhibiting PCSK9 gene expression" includes any level of inhibition of the PCSK9 gene, such as at least partial inhibition of PCSK9 gene expression, such as inhibition of 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%.

[0073] Based on any variable level associated with PCSK9 gene expression, such as PCSK9 mRNA level, PCSK9 protein level, or serum lipid level, the expression of the PCSK9 gene can be assessed. Inhibition can be assessed by the reduction of one or more of these variables in absolute or relative levels compared to a control level. A control level can be any type of control level utilized in the art, such as a baseline level before administration or a level determined from a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0074] As used herein, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals, such as monkeys. Most preferably, the subject or patient is a human.

[0075] As used herein, "PCSK9-related disease" is intended to include any disease associated with the PCSK9 gene or protein. Such diseases can be caused, for example, by overproduction of PCSK9 protein, by mutations in the PCSK9 gene, by abnormal cleavage of the PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipid disorders, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these disorders, such as heart and circulatory system diseases.

[0076] As used herein, a "therapeutically effective amount" is intended to include an amount of a RNAi agent that, when administered to a patient for the treatment of a PCSK9-associated disease, is sufficient to achieve treatment of the disease (e.g., by attenuating, ameliorating, or maintaining the existing disease or one or more disease symptoms). The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and medical history, age, weight, family history, genetic makeup, the stage of the pathological process mediated by PCSK9 expression, the type of previous or concomitant therapy (if any), and other individual characteristics of the patient to be treated.

[0077] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that is sufficient to prevent or ameliorate a PCSK9-related disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or displayed symptoms of the disease but may be susceptible to the disease. Improving a disease includes slowing the progression of the disease or reducing the severity of a subsequent disease. The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the risk level of the disease, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0078] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0079] As used herein, the term "sample" includes similar fluids, cells or tissues separated from a subject, and a collection of fluids, cells or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluid, plasma, cerebrospinal fluid, ocular fluid (ocular fluid), lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local regions. For example, samples can be derived from the fluid or cells in a particular organ, an organ part or these organs. In certain embodiments, samples can be derived from liver (for example, whole liver or some sections of liver, or some types of cells in liver, for example, hepatocytes). In a preferred embodiment, "sample derived from experimenter" refers to blood or plasma extracted from this experimenter. In other embodiments, "sample derived from experimenter" refers to liver tissue (or its subcomponent) derived from this experimenter.

[0080] In one aspect, the present disclosure provides a double-stranded RNAi agent for reducing the expression of PCSK9, comprising any one oligonucleotide duplex selected from the following sense and antisense strand pairs:

[0081] (1) The sense strand has the sequence shown in SEQ ID NO. 1 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof;

[0082] (2) the sense strand has a sequence as shown in SEQ ID NO. 2 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof;

[0083] (3) the sense strand has a sequence as shown in SEQ ID NO. 3 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence thereof;

[0084] (4) the sense strand has a sequence as shown in SEQ ID NO. 4 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence thereof;

[0085] (5) the sense strand has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified sequence thereof;

[0086] (6) the sense strand has a sequence as shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 18 or a fragment thereof, or a modified sequence thereof;

[0087] (7) the sense strand has a sequence as shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 19 or a fragment thereof, or a modified sequence thereof;

[0088] (8) The sense strand has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified sequence thereof;

[0089] (9) the sense strand has a sequence as shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 21 or a fragment thereof, or a modified sequence thereof;

[0090] (10) The sense strand has a sequence as shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 22 or a fragment thereof, or a modified sequence thereof;

[0091] (11) the sense strand has a sequence as shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 23 or a fragment thereof, or a modified sequence thereof;

[0092] (12) The sense strand has a sequence as shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 24 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0093] In some embodiments, all nucleotides on the sense and antisense strands are modified nucleotides.

[0094] In some embodiments, the double-stranded RNAi agent is an RNAi agent for inhibiting expression of the PCSK9 gene.

[0095] In some embodiments, the sense strand differs from any one of SEQ ID NOs. 1-12 by 1-3 nucleotides.

[0096] In some embodiments, the antisense strand differs from any one of SEQ ID NOs. 13-24 by 1-3 nucleotides.

[0097] In some embodiments, at least one modified nucleotide is selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0098] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0099] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0100] In some embodiments, the double-stranded region is 15-30 nucleotide pairs in length.

[0101] In some embodiments, the double-stranded region is 17-25 nucleotide pairs in length.

[0102] In some embodiments, the double-stranded region is 19-23 nucleotide pairs in length.

[0103] In some embodiments, the double-stranded region is 21 nucleotide pairs in length.

[0104] In some embodiments, each strand has 15-30 nucleotides.

[0105] In some embodiments, each strand has 19-25 nucleotides.

[0106] In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0107] In some embodiments, all nucleotide modifications on the sense strand and the antisense strand are chemical modifications of the 2' position of the ribose sugar of the nucleotide.

[0108] In some embodiments, the chemical modification at the 2' position of the ribose of the nucleotide is selected from any one or a combination of 2'-methoxy, 2'-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino and 2'-pyridylmethoxy.

[0109] In some embodiments, the chemical modification at the 2' position of the ribose sugar of each nucleotide is selected from a combination of 2'-methoxy and 2'-fluoro.

[0110] In some embodiments, the chemical modification at the 2' position of the ribose sugar of each nucleotide is selected from alternating combinations of 2'-methoxy and 2'-fluoro.

[0111] In some embodiments, the chemical modification of the 2' position of each nucleotide ribose is as follows: the odd-numbered positions of the sense chain are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified; the odd-numbered positions of the antisense chain are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified.

[0112] In some embodiments, the nucleotide monomers are linked by 3',5'-phosphodiester bonds.

[0113] In some embodiments, the 3',5'-phosphodiester bonds between nucleotide monomers are thio-modified.

[0114] In some embodiments, the aforementioned oligonucleotides have the following modifications:

[0115] The antisense strand is modified in one of the following ways:

[0116] The sense strand is modified in one of the following ways:

[0117] In the above table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone;

[0118] The antisense strand adopts modification A, and the sense strand adopts modification a;

[0119] The antisense strand adopts modification B, and the sense strand adopts modification a;

[0120] The antisense strand adopts modification C, and the sense strand adopts modification a;

[0121] The antisense strand adopts modification B, and the sense strand adopts modification b;

[0122] The antisense strand adopts modification C, and the sense strand adopts modification b.

[0123] In some embodiments, the antisense strand uses a modification group in the second to eighth positions from the 5' end, wherein the modification group is selected from UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows:

[0124] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0125] In some embodiments, the phosphorylation of the 5'-carbon atom of the 5'-terminal nucleotide glycoside of the modified antisense strand includes, but is not limited to, the following 5'-phosphorylation groups: 5'-vinylphosphonate group (5'-E-VP); 5'-methylphosphonate group (5'-MP); 5'-C-methylphosphonate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structures of which are shown below:

[0126] Wherein, R is hydrogen, hydroxyl, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 alkylcarbonylamino or halogen;

[0127] The base is selected from adenine, guanine, cytosine, thymine and uracil.

[0128] In some embodiments, the 3',5'-phosphodiester bonds connecting the nucleotide monomers at the ends of the sequences are thio-modified and form chirally pure 3',5'-phosphothioate diester bonds, wherein the 5' ends of the sense strand and the antisense strand contain 1-3 thio linkages, and the 3' end of the antisense strand contains 1-3 thio linkages.

[0129] The double-stranded RNA (dsRNA) agents of the present disclosure may optionally be conjugated to one or more ligands. The ligand may be attached to the sense strand, antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the ligand may be conjugated to the sense strand. In a preferred embodiment, the ligand is bound to the 3' end of the sense strand. In a preferred embodiment, the ligand is a GalNAc ligand.

[0130] In another aspect, the present disclosure provides a conjugate for reducing the expression of PCSK9, comprising the double-stranded RNAi agent and a ligand conjugated thereto.

[0131] In some embodiments, the ligand is conjugated to the 3'-end or the 5'-end of the sense strand of the oligonucleotide.

[0132] In some embodiments, the ligand is one or more GalNAc derivatives attached using a divalent or trivalent branched linkage.

[0133] In some embodiments, the ligand is:

[0134] wherein X is hydrogen or a hydroxyl protecting group or H, wherein the hydroxyl protecting group includes acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; and q, r and s are independently integers between 1 and 7.

[0135] In some embodiments, the ligand is:

[0136] In some embodiments, the ligand is:

[0137] Wherein, X is oxygen, nitrogen or sulfur;

[0138] Y is an alkyl group or an aromatic group;

[0139] R1 is oxygen or sulfur;

[0140] R2 is hydrogen, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy or halogen;

[0141] 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;

[0142] B is -(CH2) e -, where e is an integer from 0 to 7;

[0143] L is -CONH- or -NHCO-;

[0144] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0145] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0146] Y1 is 0 or 1;

[0147] Y2 is 0, 1, or 2;

[0148] Y3 is 1, 2 or 3;

[0149] m is an integer from 0 to 4;

[0150] n is an integer from 0 to 4.

[0151] In some embodiments, the ligand is G4, G5, G6, or G7:

[0152] In some embodiments, the conjugate has the structure shown below:

[0153] In some embodiments, the double-stranded RNAi agent comprises any one oligonucleotide duplex selected from the following sense and antisense strand pairs:

[0154] (1) 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;

[0155] (2) 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;

[0156] (3) 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;

[0157] (4) 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;

[0158] (5) 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;

[0159] (6) 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;

[0160] (7) 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;

[0161] (8) 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;

[0162] (9) 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;

[0163] (10) 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;

[0164] (11) 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;

[0165] (12) 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;

[0166] (14) 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;

[0167] (15) 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; and

[0168] (16) 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;

[0169] Wherein, the oligonucleotide duplex is conjugated with ligand G4, G5, G6 or G7.

[0170] In some embodiments, the double-stranded RNAi agent comprises any one oligonucleotide duplex selected from the following sense and antisense strand pairs:

[0171] (1) 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;

[0172] (2) 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; and

[0173] (3) 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;

[0174] Wherein, the oligonucleotide duplex is conjugated with ligand G4, G5, G6 or G7.

[0175] In some embodiments, the double-stranded RNAi agent comprises an oligonucleotide duplex consisting of a sense strand represented by SEQ ID NO. 353 and an antisense strand represented by SEQ ID NO. 448, and the oligonucleotide duplex is conjugated to a ligand G5.

[0176] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the double-stranded RNAi agent or conjugate, and a pharmaceutically acceptable carrier.

[0177] In certain embodiments, provided herein are pharmaceutical compositions containing RNAi as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing RNAi can be used to treat diseases or conditions associated with the expression or activity of PCSK9 genes, such as lipid disorders. Such pharmaceutical compositions are formulated based on delivery models. One example is a composition formulated to be delivered systemically by parenteral delivery, such as by intravenous (IV) delivery. Another example is the following composition, which is formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, such as by continuous pump infusion.

[0178] Pharmaceutical compositions comprising the RNAi agents of the present disclosure can be, for example, solutions with or without a buffer or compositions containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.

[0179] In the method of the present disclosure, the RNAi agent can be administered in a solution. A free RNAi agent can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, the free siRNA can also be administered in a suitable buffer solution. The buffer solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and volume molar osmotic pressure concentration of the buffer containing the RNAi agent can be adjusted so that it is suitable for administering to the subject.

[0180] In some embodiments, the buffer solution further comprises an agent 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 agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0181] The pharmaceutical composition of the present invention can be administered at a dosage sufficient to inhibit the expression of the PCSK9 gene. Typically, a suitable dosage of the RNAi of the present invention is in the range of about 0.001 to about 200.0 mg per kilogram of body weight of the recipient per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. For example, an RNAi agent (e.g., dsRNA) can be administered at 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, 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. .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, 48, 49, or about 50 mg / kg.

[0182] The pharmaceutical composition can be given once a day, or the RNAi can be given twice, three times or more sub-doses at appropriate intervals within one day, or even can be given by continuous infusion or delivery through a controlled release formulation. In this case, the RNAi contained in each sub-dose must be correspondingly less, so as to realize a daily total dose. Dosage unit compounding can also be used to deliver within a few days, for example, using a conventional sustained release formulation that provides a lasting RNAi release within a few days' timeframe. Slow-release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, and can be used with reagents of the present disclosure thus. In this embodiment, the dosage unit comprises a plurality of corresponding daily doses.

[0183] In other embodiments, a single dose of the pharmaceutical composition can be long-lasting, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per week. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once every two months.

[0184] Those skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and other existing diseases. In addition, treating a subject with a therapeutically effective dose of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dosage and in vivo half-life of each RNAi encompassed by the present disclosure may be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0185] The pharmaceutical compositions of the present disclosure can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a skin patch); pulmonary; e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal; intranasal; epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration.

[0186] The RNAi used in the compositions and methods of the present disclosure can be formulated for delivery in a membrane molecular assembly such as a liposome or micelle. 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 single-layer or multi-layer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi composition. The lipophilic material separates the aqueous interior from the aqueous exterior that typically does not include the RNAi composition (although in some instances, it may include it). Liposomes are useful for transferring and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when liposomes are applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the fusion of the liposome and the cell proceeds, the internal aqueous contents including the RNAi are delivered into the cell, where the RNAi can specifically bind to a target RNA and can mediate RNAi. In some cases, the liposomes are also specifically targeted, for example to direct the RNAi to a particular cell type.

[0187] Liposomes containing RNAi agents can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent formulation is then added to the micelles containing the lipid component. The cationic groups on the lipid 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.

[0188] RNAi, such as dsRNA of the present disclosure, can be fully encapsulated in a lipid formulation (eg, LNP or other nucleic acid-lipid particle).

[0189] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNP contains a cationic lipid, a non-cationic lipid, and a lipid (e.g., a PEG-lipid conjugate) that stops the particle aggregation. LNP is extremely useful for synthetic applications because they demonstrate a circulation life that is extended after intravenous (iv) injection and accumulate at distal sites (e.g., at a site physically separated from the site of administration).

[0190] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0191] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0192] In some preferred embodiments, the cationic lipid is a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12 ~ 25 Branched alkyl, for example, YK-009 of formula (II) (see patent CN114044741B).

[0193] In some preferred embodiments, the cationic lipid is a compound of formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein: G1 is C 2～8 Alkylene; G2 is C 2～8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6～25 Straight or branched alkyl; R2 is C 6～25 A linear 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-. For example, YK-401 of formula (II-I) and YK-402 of formula (II-II) (see patent CN115784921B).

[0194] In some preferred embodiments, the cationic lipid is a compound of formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein: G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～20 Straight or branched alkyl; R2 is C12～25 Branched alkyl group; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(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-. For example, YK-201 of formula (III-I) and YK-202 of formula (III-II) (see patent CN115677518B).

[0195] In some preferred embodiments, the cationic lipid is a compound of formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1～8 Alkylene; G2 is C 2～8 Alkylene; R1 is C 6～25 Straight or branched alkyl; R2 is C 12～25 Straight-chain or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. For example, YK-305 of formula (IV-I) and YK-310 of formula (IV-II) (see patent CN115745820B).

[0196] In some preferred embodiments, the cationic lipid is a compound of formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 and G 2 Each independently is an unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 hydrocarbon group; and R5 is H or a C1-C6 hydrocarbon group; for example, ALC0315 of formula (VI) (see patent CN108368028B);

[0197] In some preferred embodiments, the cationic lipid is a compound of formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R4 is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of: -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2 , -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R) )S(O)2R8 and heterocycle; n is 1, 2 or 3; each R is independently selected from the group consisting of: C1-3 alkyl, C2-3 alkenyl, (CH2)qOR* and H, and each q is independently selected from 1, 2 and 3, each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each X is independently selected from the group consisting of: F, Cl, Br and I; for example, SM102 of formula (VI-I) (see patent CN110520409A).

[0198] In some preferred embodiments, the cationic lipid is a compound of formula (VII), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof (see CN102625696B, DLIN-MC3-DMA),

[0199] In some more preferred embodiments, the cationic lipids include YK-009, YK-401, YK-305, ALC0315, SM102, and DLIN-MC3-DMA.

[0200] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0201] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0202] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0203] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0204] In some more preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0205] In some preferred embodiments, the neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0206] In some more preferred embodiments, the neutral lipid is selected from one or more of the following: 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-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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 (ME 16.0PE), 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-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0207] In some more preferred embodiments, the neutral lipid is DOPE and / or DSPC.

[0208] In some preferred embodiments, the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0209] In some more preferred embodiments, the structured lipid is cholesterol.

[0210] In some preferred embodiments, the polymer-conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0211] In some more preferred embodiments, the polymer-conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0212] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating liver disorders (e.g., liver cancer).

[0213] The pharmaceutical formulations of the present disclosure (which may conveniently be in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the steps of combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally speaking, the formulations are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier or both, and, if desired, shaping the product.

[0214] Compositions of the present disclosure can be formulated into any one of many possible dosage forms, such as but not limited to tablets, capsules, gel capsules, liquid syrups, soft capsules, suppositories and enemas. Compositions of the present disclosure can also be formulated into suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further comprise materials that increase the viscosity of the suspension, such materials including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also comprise a stabilizing agent.

[0215] Some compositions of the present disclosure also incorporate carrier compounds into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analogue, which is inert (i.e., not biologically active per se), but is considered to be a nucleic acid during in vivo processes, such as by degrading biologically active nucleic acids or promoting their removal from circulation to reduce the bioavailability of biologically active nucleic acids. Co-administration of nucleic acids and carrier compounds (generally an excess of the latter substance) can result in a significant reduction in the amount of nucleic acid recovered in the liver, kidneys, or other external circulation reservoirs, assuming that this is due to competition between the carrier compound and the nucleic acid for common receptors. For example, when co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid, the recovery of partially phosphorothioated dsRNA in liver tissue can be reduced.

[0216] In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a particular pharmaceutical composition, with reference to the intended mode of administration. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0217] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration, which do not react toxicly with nucleic acids, can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

[0218] The preparation that is used for local administration of nucleic acid can comprise the aseptic or non-sterile aqueous solution, the non-aqueous solution in common solvent such as alcohol, or the nucleic acid solution in liquid or solid oil matrix.These solutions can also comprise buffer, diluent and other suitable additives.Can use be suitable for non-parenteral administration and not with nucleic acid generation toxic reaction, pharmaceutically acceptable organic or inorganic excipient.

[0219] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0220] The dosage form, carrier compound, pharmaceutical carrier, excipient, etc. of the above composition are described in U.S. Patent No. 10125369B2, which is incorporated herein by reference.

[0221] The present disclosure also provides methods for treating or preventing diseases and conditions that can be regulated by downregulating PCSK9 gene expression. For example, the RNAi agents described herein can be used to treat lipid disorders, such as hyperlipidemia, and other forms of lipid imbalance, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these disorders, such as heart and circulatory system diseases. Other diseases and conditions that can be regulated by downregulating PCSK9 gene expression include lysosomal storage diseases, including but not limited to 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 heart disease (CHD), cerebrovascular disease (CVD), aortic valve stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), transient ischemic attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular disease and / or congestive heart failure or any other trait. The methods include administering to the subject a therapeutically effective amount or a prophylactically effective amount of a RNAi agent, conjugate or composition of the present disclosure. In some embodiments, the method includes administering a therapeutic amount of PCSK9 siRNA to a patient with a heterozygous LDLR genotype.

[0222] The RNAi agents of the present invention can be administered to a subject using any mode of administration known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, the agents are administered subcutaneously.

[0223] In other embodiments, the siRNA is administered in combination with another therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0224] Examples of other therapeutic agents include drugs known to treat lipid disorders such as hypercholesterolemia, atherosclerosis, dyslipidemia, or cardiovascular and cerebrovascular diseases. For example, other drugs for the treatment of hyperlipidemia are selected from fibrates, statins, bile acid sequestrants, and nicotinic acids. Other drugs for the treatment of 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 hydrochlorothiazide, olmesartan medoxomil, etc.), beta-blockers (e.g., propranolol, bisoprolol, metoprolol tartrate, metoprolol succinate, etc.).

[0225] In some embodiments, the RNAi agent is administered to a patient and subsequently the additional therapeutic agent is administered to the patient (or vice versa). In other embodiments, the RNAi agent and the additional therapeutic agent are administered simultaneously.

[0226] In another aspect, the present disclosure provides use of the aforementioned double-stranded RNAi agent, conjugate, or composition in the preparation of a drug for a disease associated with PCSK9.

[0227] In another aspect, the present disclosure provides use of the aforementioned double-stranded RNAi agent, conjugate, or composition in the preparation of a drug for treating hypercholesterolemia, atherosclerosis, dyslipidemia, or cardiovascular and cerebrovascular diseases.

[0228] In some embodiments, the PCSK9-related disease is selected from hypercholesterolemia, atherosclerosis, dyslipidemia or cardiovascular and cerebrovascular diseases.

[0229] 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.

[0230] The nucleotide abbreviations herein are as follows:

[0231] A = adenosine 3'-phosphate

[0232] Am=2'-O-methoxyadenosine-3'-phosphate

[0233] Ams = 2'-O-methoxyadenosine-3'-phosphorothioate

[0234] Af = 2'-fluoroadenosine-3'-phosphate

[0235] Afs = 2'-fluoroadenosine-3'-phosphorothioate

[0236] G = guanosine 3'-phosphate

[0237] Gm=2'-O-methoxyguanosine-3'-phosphate

[0238] Gms = 2'-O-methoxyguanosine-3'-phosphorothioate

[0239] Gf = 2'-fluoroguanosine-3'-phosphate

[0240] Gfs = 2'-fluoroguanosine-3'-phosphorothioate

[0241] C = cytidine-3'-phosphate

[0242] Cm=2'-O-methoxycytidine-3'-phosphate

[0243] Cms = 2'-O-methoxycytidine-3'-phosphorothioate

[0244] Cf = 2'-fluorocytidine-3'-phosphate

[0245] Cfs = 2'-fluorocytidine-3'-phosphorothioate

[0246] U = uridine-3'-phosphate

[0247] Um = 2'-O-methoxyuridine-3'-phosphate

[0248] Ums = 2'-O-methoxyuridine-3'-phosphorothioate

[0249] Uf = 2'-fluorouridine-3'-phosphate

[0250] Ufs = 2'-fluorouridine-3'-phosphorothioate

[0251] AmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyadenosine-3'-phosphorothioate

[0252] UmsEVP = 5'-vinyl-(E)-phosphonate-2'-methoxyuridine-3'-phosphorothioate

[0253] Agna = adenosine diol nucleic acid

[0254] Cgna = cytidine-diol nucleic acid

[0255] Ggna = guanosine diol nucleic acid

[0256] Tgna = thymidine diol nucleic acid

[0257] Ugna = uridine diol nucleic acid

[0258] Example

[0259] Example 1: Synthesis of alternatively modified small interfering oligonucleotides

[0260] 84 siRNA base sequences were designed based on the PCSK9 mRNA sequence. To enhance the inhibitory efficiency and stability of the sequences, the base sequences were alternatingly modified with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) groups, and the termini were thiolated. The sense strand had 2'-F modifications at all odd-numbered positions and 2'-OMe modifications at all even-numbered positions; the antisense strand had 2'-OMe modifications at all odd-numbered positions and 2'-F modifications at all even-numbered positions. Furthermore, the sense strand had two thiolation modifications at the 5' end, and the antisense strand had two thiolation modifications at both the 5' and 3' ends. The alternatingly modified siRNA sequences are shown in Table 2.

[0261] 1. Synthesis of the Alternately Modified Sequence P92-si5

[0262] The basic sequence of the small interfering RNA numbered P92-si5 in Table 2 is:

[0263] Sense strand: 5'-AAGAUCCUGCAUGUCUUCCAU-3' (SEQ ID NO. 1)

[0264] Antisense strand: 5'-AUGGAAGACAUGCAGGAUCUUGG-3' (SEQ ID NO. 13)

[0265] Odd-numbered sites on the sense strand and even-numbered sites on the antisense strand are modified with 2'-F, while all other sites are modified with 2'-OMe. Additionally, there are two thiolate modifications at the 5' end of the sense strand and two thiolate modifications at the 5' and 3' ends of the antisense strand.

[0266] Instruments and reagents: Qingke 192P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model number is Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0267] Preparation method:

[0268] The following nucleotide monomer solutions were prepared using acetonitrile at a monomer concentration of 0.15 M: 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).

[0269] Prepared by the following steps:

[0270] (1) Deprotection

[0271] A 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0272] (2) Coupling

[0273] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by rinsing with acetonitrile.

[0274] (3) Oxidation / sulfurization

[0275] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[0276] Sulfurization: 3% hydrogenated xanthan gum in pyridine was used as a sulfurizing agent for sulfurization, followed by rinsing with acetonitrile.

[0277] (4) Hydroxyl protection

[0278] Hydroxyl group protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) and tetrahydrofuran / pyridine / nitromethylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl group protecting reagents, followed by rinsing with acetonitrile.

[0279] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0280] (5) Use 3% dichloroacetic acid in toluene as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile to wash.

[0281] (6) Ammonolysis and purification

[0282] The solid phase carrier is transferred to a reactor, concentrated ammonia water (25-28%) is added, and after maintaining aminolysis at 60°C for 12 hours, the system is cooled to room temperature and the mixture is transferred to a filter press. It is eluted with a mixed solution of purified water and ethanol, the filtrate is combined, passed through a chromatography column, concentrated, and freeze-dried to obtain the product.

[0283] (7) Annealing

[0284] The purified sense chain and antisense chain 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 chain.

[0285] P92-si5 purity 97.4%; measured molecular weight: 14493.52

[0286] 2. Synthesis of other sequences

[0287] The other sequences listed in Table 2 were synthesized according to the above method.

[0288] Table 2 Alternative modified small interfering RNA sequences

[0289] Example 2: Inhibitory effect of alternating modified sequences on the PCSK9 gene

[0290] After the 2'-OMe and 2'-F alternating modified siRNA sequences synthesized in Example 1 were transfected into HELA cells via lipid nanoparticles (LNP), the inhibitory effect of each sequence on the PCSK9 gene was detected using qPCR technology.

[0291] 1. Experimental Materials

[0292] Test sample: the alternatively modified small interfering RNA sequence P92-si1-84 listed in Table 2 (synthesized in Example 1).

[0293] Cell type: HeLa cell line HELA cells

[0294] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0295] 2. Experimental Methods

[0296] qRT-PCR was used to detect the inhibitory effect of the samples on the expression of PCSK9 gene mRNA in HELA cell lines.

[0297] 2.1 Cell culture

[0298] Take subcultured HELA cells and culture them in RPMI 1640 medium (10% fetal bovine serum supplemented with 100% penicillin and streptomycin at 10 μL / mL) at logarithmically growing levels in a 37°C, 5% CO2 incubator. Change the medium daily. Digest and subculture with 0.25% trypsin. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh medium for subculture.

[0299] 2.2 Cell transfection

[0300] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 1.5:98.5 and vortex to mix thoroughly.

[0301] Prepare transfection reagent: Add 60 μL of siRNA solution diluted in Opti-MEM to 60 μL of transfection mixture at a 1:1 (v / v) ratio. Vortex to mix thoroughly and let stand at room temperature for 15 minutes to generate lipid nanoparticles (LNPs). 12.5 μL of the mixture was used for encapsulation efficiency testing.

[0302] For the blank control group, add 60 μL of the prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[0303] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA concentration in each well 1 nM. Add 500 μL of cell suspension (6×10 cells per ml) 4 After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell incubator and cultured for 40 h.

[0304] 2.3 PCSK9 mRNA detection

[0305] 1) RNA extraction

[0306] a. Aspirate the culture medium from the 12-well plate and wash the cells by adding 0.5 mL of 1× PBS to each well. Aspirate the PBS. Add 0.5 mL of TRIzol reagent to each well and thoroughly lyse the cells by pipetting. Transfer the cells to a 1.5 mL RNase-free EP tube and let them stand at room temperature for 5 minutes.

[0307] b. Add 0.1 mL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 4°C, 12,000 × g for 15 minutes, and transfer 200 μL of the supernatant to a new EP tube.

[0308] c. Add an equal volume of isopropanol and gently mix the contents by inverting the tube. Incubate at -20°C for 10 min. Centrifuge at 4°C at 12,000 × g for 15 min and discard the supernatant.

[0309] d. Add 0.5 mL of 75% ethanol to gently wash the RNA pellet. Centrifuge at 12,000 × g at 4°C for 5 minutes, and aspirate the supernatant. Repeat the rinse cycle once more, centrifuging at 12,000 × g at 4°C for 1 minute. Remove any remaining ethanol using a micropipette.

[0310] e. Allow the remaining ethanol to dry at room temperature for 2-3 minutes, then add 40 μL of RNase-free ddH2O to dissolve.

[0311] 2) RNA concentration detection

[0312] RNA concentration was measured using a nanodrop. A blank control of 2 μL of RNase-free ddH2O was used, and 2 μL of RNA sample was used for each test. The sample concentration was recorded.

[0313] 3) PCSK9 mRNA quantitative detection

[0314] In a 15 mL centrifuge tube, add the remaining components except primers and template in 7.5×84=630 portions, marked as A.

[0315] Label 1.5 mL EP tubes (84 pieces), add 77 μL of A and 420 ng of total RNA to each tube, mix well and set aside (labeled as B).

[0316] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 were mixed and set aside (marked as C).

[0317] Add 1 μL of B1 and 1 μL of C to each well of the PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and transfer to the microcentrifuge.

[0318] *This step is performed on ice to maintain low temperature conditions.

[0319] Place the plate in the qPCR instrument and run the following program:

[0320] Reverse transcription: 55°C, 15 min;

[0321] Pre-denaturation: 95°C, 30 sec;

[0322] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0323] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0324] The run time was approximately 2 h. Analyze the experimental results and calculate 2-ΔΔCt.

[0325] 2.4 Data Processing

[0326] The calculation formula for PCSK9 mRNA expression rate (%) is:

[0327] Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) × 100%;

[0328] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0329] 3. Experimental Results

[0330] The inhibition rate in this example is the average of four experiments. The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4-6.

[0331] Experimental results showed that some sequences modified with alternating 2'-OMe and 2'-F, 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 (see Table 3 for specific sequences), significantly inhibited 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 exceeding 70%.

[0332] The other sequences had poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates lower than 50%.

[0333] Table 3 siRNA sequences with significant inhibitory effects on PCSK9 gene

[0334] The inhibition rate of each sequence on PCSK9 mRNA expression in HELA cells is shown in Tables 4-6.

[0335] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, 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. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[0336] Table 4 Alternative modified sequences with significant inhibitory effect on PCSK9 gene (inhibition rate higher than 50%)

[0337] As can be seen from Table 4, the alternatively 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 (see Table 3 for specific sequences), a total of 12 sequences, have a significant inhibitory effect on PCSK9 mRNA expression, with inhibition rates of more than 50%. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70% (Figure 1). These 12 sequences can be used as candidate sequences.

[0338] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0339] Table 5 Alternative modified sequences with 30%-50% inhibition rate on PCSK9 gene

[0340] The 26 sequences listed in Table 5 had poor inhibitory effects on the PCSK9 gene, with inhibition rates below 50%, 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).

[0341] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0342] Table 6 Alternative modified sequences with PCSK9 gene inhibition rate below 30%

[0343] The 46 sequences listed in Table 6 had very poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates all below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively (Figures 3 and 4).

[0344] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 is 61.8% higher than that of P92-si4, which is a significant improvement.

[0345] The comparison of the inhibitory effects of some siRNAs with similar sequences on PCSK9 mRNA is shown in Table 7.

[0346] Table 7 Comparison of the inhibition rate of PCSK9 gene expression by siRNAs with similar sequences

[0347] As can be seen from Table 7, some siRNAs with similar sequences have very different inhibitory effects on PCSK9 mRNA expression.

[0348] Base sequence 4 differs from base sequence 5 only by one base at the end. The sense strand has an additional C at the 5' end of base sequence 4, and an additional U at the 3' end of base sequence 5; the rest of the sequences are identical. The antisense strand has an additional U at the 3' end of base sequence 4, and an additional A at the 5' end of base sequence 5; the rest of the sequences are identical. However, the inhibition rate of the alternatively modified sequence P92-si5 was significantly higher, increasing by 61.8% compared to P92-si4.

[0349] Base sequences 50 and 51 differ only in the terminal six bases. The sense strand has GAUUAA at the 5' end and CUGGAU at the 3' end of 50, with the rest of the sequence identical. The antisense strand has AAUCAG at the 3' end and AUCCAG at the 5' end of 51, with the rest of the sequence identical. However, the inhibition rate of the alternatively modified sequence P92-si51 was significantly higher, reaching 46.8% compared to P92-si50.

[0350] Base sequence 2 differs from base sequence 3 only in the terminal six bases. The sense strand: base sequence 2 has a 5' end of AUACCU, while base sequence 3 has a 3' end of UGUCUU; the rest of the sequence is identical. The antisense strand: base sequence 2 has a 3' end of GUAUCC, while base sequence 3 has a 5' end of AAGACA; the rest of the sequence is identical. However, the inhibition rate of the alternatively modified sequence P92-si3 was significantly higher, by 45.9%, than that of P92-si2.

[0351] Base sequences 9 and 10 differ only in their terminal two bases. The sense strand of base sequence 9 has a GG at its 5' end, while base sequence 10 has an AG at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 9 has a UA at its 3' end, while base sequence 10 has a CU at its 5' end; the rest of the sequence is identical. However, the inhibition rate of the alternating sequence P92-si9 was significantly higher, increasing by 53.1% compared to that of P92-si10.

[0352] Base sequence 21 differs from base sequence 22 only at the two terminal bases. The sense strand has a CA at the 5' end of base sequence 21 and a GA at the 3' end of base sequence 22, with the remaining sequences identical. The antisense strand has a CA at the 3' end of base sequence 21 and a UC at the 5' end of base sequence 22, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si21 was significantly higher, increasing by 65.6% compared to P92-si22.

[0353] Base sequences 30 and 31 differ only in one base at the end. The sense strand has an A at the 5' end of base sequence 30 and a G at the 3' end of base sequence 31, with the remaining sequences identical. The antisense strand has a G at the 3' end of base sequence 30 and a C at the 5' end of base sequence 31, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si31 was significantly higher, by 39.4%, compared to P92-si30.

[0354] Base sequences 73 and 74 differ only in one terminal base. The sense strand has a G at the 5' end of base sequence 73, while the 3' end of base sequence 74 has a G, with the remaining sequences identical. The antisense strand has an A at the 3' end of base sequence 73, while the 5' end of base sequence 74 has a C, with the remaining sequences identical. However, the inhibition rate of the alternatively modified sequence P92-si73 was significantly higher, increasing by 62.2% compared to P92-si74.

[0355] Therefore, it is not easy to screen out sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and it requires a lot of creative work.

[0356] Summarize:

[0357] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, 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. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[0358] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0359] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0360] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 was 61.8% higher than that of P92-si4, which is a significant improvement. Therefore, it is not easy to screen out sequences with significant inhibitory activity from a large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and a lot of creative work is required.

[0361] Example 3: Inhibitory effect of unmodified sequences on the PCSK9 gene

[0362] In this example, some 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 detected using qPCR technology, and unmodified siRNA sequences with better inhibitory effects were screened out.

[0363] 1. Experimental Materials

[0364] Test samples:

[0365] The unmodified small interfering RNA sequences are listed in Table 8. All sequences were synthesized according to the method in Example 1.

[0366] Table 8 Unmodified siRNA sequences

[0367] Cell type: HeLa cell line HELA cells

[0368] Drug solvent: sterile enzyme-free water, Gibco Opti-MEM.

[0369] 2. Experimental Methods

[0370] Refer to Example 2.

[0371] 3. Experimental Results

[0372] The inhibition rates of PCSK9 mRNA expression by each unmodified sequence are shown in Tables 9 and 10.

[0373] The unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73 and si83 had significant inhibitory effects on the expression of PCSK9 mRNA in HELA cells, with inhibition rates all above 50%, among which the inhibition rates of si5, si81, si82, si3 and si8 exceeded 60%.

[0374] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[0375] Table 9 Unmodified sequences with significant inhibitory effect on PCSK9 gene (inhibition rate greater than 50%)

[0376] The unmodified sequences listed in Table 9, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, significantly inhibited PCSK9 mRNA expression, with inhibition rates exceeding 50%. Among them, si5, si81, si82, si3, and si8 exhibited inhibition rates exceeding 60% ( FIG5 ). These 12 sequences can be used as candidate sequences.

[0377] (2) The inhibition rates of other unmodified sequences on the PCSK9 gene were all lower than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[0378] Table 10: Unmodified sequences with an inhibition rate of less than 40% on the PCSK9 gene

[0379] The sequences in Table 10 had very poor inhibitory effects on PCSK9 mRNA expression, with inhibition rates all below 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% ( FIG. 6 ).

[0380] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[0381] The comparison of the inhibitory effects of some siRNAs with similar sequences on PCSK9 mRNA is shown in Table 11.

[0382] Table 11 Comparison of the inhibition rate of PCSK9 gene expression by unmodified sequences with similar sequences

[0383] As can be seen from Table 11, some siRNAs with similar sequences have very different inhibitory effects on PCSK9 mRNA expression.

[0384] Base sequence 4 differs from base sequence 5 only by one base at the end. The sense strand has an additional C at the 5' end of base sequence 4, while the 3' end of base sequence 5 has an additional U; the rest of the sequences are identical. The antisense strand has an additional U at the 3' end of base sequence 4, while the 5' end of base sequence 5 has an additional A; the rest of the sequences are identical. However, base sequence 5 significantly increases its inhibition rate by 51.2% compared to base sequence 4.

[0385] Base sequence 50 differs from base sequence 51 only in the terminal six bases. The sense strand of base sequence 50 has a 5' end of GAUUAA, while the 3' end of base sequence 51 has a CUGGAU, with the rest of the sequence identical. The antisense strand of base sequence 50 has a 3' end of AAUCAG, while the 5' end of base sequence 51 has a 5' end of AUCCAG, with the rest of the sequence identical. However, the inhibition rate of base sequence 51 was significantly higher, by 42.6%, compared to base sequence 50.

[0386] Base sequence 2 differs from base sequence 3 only in the terminal six bases. The sense strand: base sequence 2 has a 5' end of AUACCU, while base sequence 3 has a 3' end of UGUCUU; the rest of the sequence is identical. The antisense strand: base sequence 2 has a 3' end of GUAUCC, while base sequence 3 has a 5' end of AAGACA; the rest of the sequence is identical. However, base sequence 3 significantly increased its inhibition rate by 42.7% compared to base sequence 2.

[0387] Base sequences 9 and 10 differ only in their terminal two bases. The sense strand of base sequence 9 has a GG at its 5' end, while base sequence 10 has an AG at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 9 has a UA at its 3' end, while base sequence 10 has a CU at its 5' end; the rest of the sequence is identical. However, the inhibition rate of base sequence 9 was significantly higher, by 41.2%, compared to base sequence 10.

[0388] Base sequence 21 differs from base sequence 22 only in the two terminal bases. The sense strand of base sequence 21 has a CA at its 5' end, while the 3' end of base sequence 22 has a GA, with the remaining sequences identical. The antisense strand of base sequence 21 has a CA at its 3' end, while the 5' end of base sequence 22 has a UC, with the remaining sequences identical. However, the inhibition rate of base sequence 21 was significantly higher, by 53.2%, compared to base sequence 22.

[0389] Base sequence 30 differs from base sequence 31 only at the terminal end. The sense strand of base sequence 30 has an A at its 5' end, while base sequence 31 has a G at its 3' end; the rest of the sequences are identical. The antisense strand of base sequence 30 has a G at its 3' end, while base sequence 31 has a C at its 5' end; the rest of the sequences are identical. However, base sequence 31 significantly increased its inhibition rate by 32.5% compared to base sequence 30.

[0390] Base sequence 73 differs from base sequence 74 only by a single terminal base. The sense strand of base sequence 73 has a G at its 5' end, while base sequence 74 has a G at its 3' end; the rest of the sequence is identical. The antisense strand of base sequence 73 has an A at its 3' end, while base sequence 74 has a C at its 5' end; the rest of the sequence is identical. However, the inhibition rate of base sequence 73 was significantly higher, by 50.2%, compared to base sequence 74.

[0391] Therefore, it is not easy to screen out sequences with significant inhibitory activity from a very large number of oligonucleotide sequences designed against the PCSK9 mRNA sequence, and it requires a lot of creative work.

[0392] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[0393] Table 12 Comparison of the inhibition rate of PCSK9 gene by alternating modified sequences and unmodified sequences

[0394] As can be seen from Table 12, the effects of alternating modifications on PCSK9 mRNA inhibition rates were inconsistent across different sequences. Some showed significant improvements, such as a 12.4% increase in inhibition rate for base sequence 5 alternating modification compared to the unmodified sequence, and a 12.2% increase in inhibition rate for base sequence 82 alternating modification compared to the unmodified sequence. Other improvements were less significant, such as the inhibition rates for base sequences 4, 22, and 52 alternating modification sequences compared to the unmodified sequence, which showed essentially no difference.

[0395] Therefore, not all unmodified sequences can have their activity improved after alternating modification, and the effects of alternating modification on the activities of different sequences are not consistent.

[0396] Summarize:

[0397] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[0398] (2) The inhibition rate of other unmodified sequences on the PCSK9 gene was less than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[0399] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[0400] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[0401] Example 4: Inhibition of PCSK9 gene using template-modified sequences

[0402] In this example, 12 sequences, namely the unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83, screened in Example 3, were modified using a modification template. DV25, DV26, DV27, DV28, DV29, DV30, and DV310 are new modification templates designed in this disclosure, and DV21 and DV22 are previously published Advanced ESC modification templates.

[0403] 1. Experimental Materials

[0404] Test samples:

[0405] Table 13 lists the sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73 and si83 in Example 3 modified using different templates (DV25, DV26, DV27, DV28, DV29, DV30, DV31, DV21 and DV22), and the corresponding alternatively 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).

[0406] The modification principles of the disclosed modified template are as follows:

[0407] DV25-29:

[0408] The antisense strand is modified in one of the following ways:

[0409] The sense strand is modified in one of the following ways:

[0410] In the above table, PS is a phosphorothioate skeleton.

[0411] The siRNA modified template with modification method A on the antisense strand and modification method a on the sense strand was named DV25;

[0412] The siRNA modified template with modification method B on the antisense strand and modification method a on the sense strand was named DV26;

[0413] The siRNA modified template with modification method C on the antisense strand and modification method a on the sense strand was named DV27;

[0414] The siRNA modification template with the antisense strand modified by method B and the sense strand modified by method b was named DV28;

[0415] The siRNA modified template with modification method C on the antisense strand and modification method b on the sense strand was named DV29.

[0416] DV30:

[0417] Antisense strand:

[0418] Chain of Justice:

[0419] In the above table, PS is a phosphorothioate skeleton.

[0420] DV31:

[0421] Antisense strand:

[0422] Chain of Justice:

[0423] In the above table, PS is a phosphorothioate skeleton.

[0424] DV21:

[0425] Antisense strand:

[0426] Chain of Justice:

[0427] In the above table, PS is a phosphorothioate skeleton.

[0428] DV22:

[0429] Antisense strand:

[0430] Chain of Justice:

[0431] In the above table, PS is a phosphorothioate skeleton.

[0432] The modified sequences of each template are detailed in Table 13, and the synthesis methods of each sequence are the same as in Example 1.

[0433] Table 13 Sequences modified using different modification templates

[0434] Cell type: HEP3B cell line

[0435] Drug solvent: sterile enzyme-free water, Gibco DMEM (purchased from Thermo Fisher Scientific; catalog number: 10569010)

[0436] 2. Experimental Methods

[0437] qRT-PCR was used to detect the inhibitory effect of the test samples on PCSK9 mRNA expression in the HEP3B cell line. Hep3B cells were provided by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. (ATCC-tings-1618164).

[0438] 2.1 Cell culture

[0439] Take the subcultured HEP3B cell line and culture the exponentially growing cells in RPMI 1640 medium (supplemented with 100 μL / mL each of penicillin and streptomycin) with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Change the medium daily. Digest and subculture with 0.25% trypsin. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add fresh medium for subculture.

[0440] 2.2 Cell transfection

[0441] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM at a ratio of 1.5:98.5 and vortex to mix thoroughly.

[0442] Prepare transfection reagent: Add 60 μL of siRNA solution diluted in Opti-MEM to 60 μL of transfection mixture at a 1:1 (v / v) ratio. Vortex to mix thoroughly and let stand at room temperature for 15 minutes to generate lipid nanoparticles (LNPs). 12.5 μL of the mixture was used for encapsulation efficiency testing.

[0443] For the blank control group, add 60 μL of the prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix thoroughly and let stand at room temperature for 15 minutes.

[0444] Add the prepared transfection reagent to a 24-well cell culture plate (100 μL per well) to make the final siRNA concentration in each well 0.05 nM. Add 500 μL of cell suspension (6×10 cells per ml) 4 After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell incubator and cultured for 40 h.

[0445] 2.3 PCSK9 mRNA detection

[0446] The steps are the same as those in “2.3 PCSK9 mRNA detection” in Example 2.

[0447] 2.4 Data Processing

[0448] The calculation formula for PCSK9 mRNA expression rate (%) is:

[0449] Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) × 100%;

[0450] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0451] 2.5 EC50 experiment

[0452] In this experiment, the siRNA concentration for the EC50 experiment of each sequence was selected from 0.2nM, 4-fold dilution, and a total of 8 concentration points (0.2nM, 0.05nM, 0.0125nM, 3.13pM, 0.78pM, 0.2pM, 0.05pM and 0.01pM). The inhibition rate of each sequence at each concentration was measured, and a graph was drawn to calculate the EC50 concentration of each sequence.

[0453] 3. Experimental Results

[0454] After three repeated experiments, the inhibition rates of each modified sequence on the PCSK9 gene in Hep3B cells are shown in Tables 14-25.

[0455] Activity assay results showed that 12 candidate sequences (unmodified sequences si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83) modified with the disclosed template DV25-29 exhibited significant inhibition against the PCSK9 gene, with inhibition rates exceeding 70%. Among them, P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 achieved inhibition rates of 89.3%, 89.3%, and 89.1%, respectively.

[0456] Using the disclosed modified template DV25-29 sequence significantly improved the inhibition of PCSK9 gene expression compared to the alternating modified sequence. For example, the inhibition rate of base sequence 51 using the template DV27 modified sequence P92-si51-DV27 increased by 29.1% compared to the alternating modified sequence, and the inhibition rate of base sequence 81 using the template DV26 modified sequence P92-si81-DV26 increased by 26.9% compared to the alternating modified sequence.

[0457] Furthermore, the activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 51 was modified with the disclosed template DV27, the inhibition rate increased by 22.8% compared to when it was modified with the disclosed template DV31. Furthermore, the inhibition rate increased by 21.3% compared to when it was modified with the previously disclosed Advanced ESC template DV21.

[0458] EC50 experiments showed that sequences modified using the disclosed design templates exhibited EC50 values ​​ranging from 0.0001 to 0.005 nM. For example, the EC50 values ​​for P92-si81-DV27, P92-si84-DV26, and P92-si82-DV27 were 0.0003 nM, 0.0004 nM, and 0.0006 nM, respectively. These sequences effectively inhibited PCSK9 gene expression at low concentrations.

[0459] (i) Effect of template modification on PCSK9 gene inhibition of each basic sequence

[0460] (1) Basic sequence 5

[0461] Table 14 PCSK9 gene inhibition rate after template modification of basic sequence 5

[0462] I. DV25-29 template of the present disclosure

[0463] Modification of base sequence 5 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 80%, significantly improving PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV26 and DV27 increased the inhibition rates of P92-si5-DV26 and P92-si5-DV27 by 19.5% and 16.6%, respectively (Figure 7).

[0464] II. Other modified templates disclosed herein

[0465] After the basic sequence 5 was modified with templates DV30 and DV31, the inhibition rates of P92-si5-DV30 and P92-si5-DV31 on the PCSK9 gene were 65.3% and 62.4%, respectively, which were 0.1% and 3.0% lower than those of the corresponding alternatively modified sequences.

[0466] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si5-DV26 and P92-si5-DV27 were significantly improved by 19.6% and 16.7% compared to P92-si5-DV30, and by 22.5% and 19.6% compared to P92-si5-DV31, respectively.

[0467] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0468] After the basic sequence 5 was modified with templates DV21 and DV22, the inhibition rates of P92-si5-DV21 and P92-si5-DV22 on the PCSK9 gene were 68.2% and 69.1%, respectively, which were 2.8% and 3.7% higher than those of the alternatively modified sequences.

[0469] Using the modified templates DV26 and DV27 of the present disclosure to modify the sequences, the inhibition rates of P92-si5-DV26 and P92-si5-DV27 were significantly improved by 16.7% and 13.8% compared with P92-si5-DV21, and by 15.8% and 12.9% compared with P92-si5-DV22, respectively.

[0470] From this we can see that:

[0471] 1) Base sequence 5, modified with the disclosed template DV25-29, exhibited significantly enhanced PCSK9 gene inhibition compared to the alternating modified sequence. For example, base sequence 5, modified with DV26, exhibited a 19.5% higher inhibition rate than the alternating modified sequence.

[0472] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 5 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 22.5% compared to when modified with the disclosed templates DV30 and DV31. Furthermore, when modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 16.7%.

[0473] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0474] (2) Basic sequence 51

[0475] Table 15 PCSK9 gene inhibition rate after template modification of basic sequence 51

[0476] I. DV25-29 template of the present disclosure

[0477] After base sequence 51 was modified with the disclosed templates DV25-29, the inhibition rates against the PCSK9 gene exceeded 75%. This was significantly improved compared to modifications with alternating 2'-methoxy and 2'-fluoro groups. For example, after modification with templates DV26 and DV27, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 increased by 27.7% and 29.1%, respectively.

[0478] II. Other modified templates disclosed herein

[0479] After the base sequence 51 was modified with templates DV30 and DV31, the inhibition rates of P92-si51-DV30 and P92-si51-DV31 on the PCSK9 gene were 63.5% and 62.8%, respectively, which were only 7.0% and 6.3% higher than those of the corresponding alternatively modified sequences.

[0480] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 were significantly improved by 20.7% and 22.1% compared with P92-si51-DV30, and by 21.4% and 22.8% compared with P92-si51-DV31, respectively.

[0481] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0482] After the basic sequence 51 was modified with the published Advanced ESC templates DV21 and DV22, the inhibition rates of P92-si51-DV21 and P92-si51-DV22 on the PCSK9 gene were 64.3% and 65.1%, respectively, which were 7.8% and 8.6% higher than those of the alternatively modified sequences.

[0483] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si51-DV26 and P92-si51-DV27 were significantly improved by 19.9% ​​and 21.3% compared with P92-si51-DV21, and by 19.1% and 20.5% compared with P92-si5-DV22, respectively.

[0484] From this we can see that:

[0485] 1) Base sequence 51, modified with the disclosed template DV25-29, significantly improved PCSK9 gene inhibition compared to the alternating modified sequence. For example, base sequence 51, modified with DV27, showed a 29.1% higher inhibition rate than the alternating modified sequence.

[0486] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 51 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 22.8% compared to when modified with the disclosed templates DV30 and DV31. Furthermore, when modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 21.3%.

[0487] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0488] (3) Basic sequence 81

[0489] Table 16 PCSK9 gene inhibition rate after template modification of basic sequence 81

[0490] I. Modification using the disclosed DV25-29 template

[0491] Modification of base sequence 81 with the disclosed templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 80%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV26 and DV27 increased the inhibition rates of P92-si81-DV26 and P92-si81-DV27 by 26.9% and 24.7%, respectively.

[0492] II. Other modified templates disclosed herein

[0493] After the basic sequence 81 was modified with templates DV30 and DV31, the inhibition rates of P92-si81-DV30 and P92-si81-DV31 on the PCSK9 gene were 71.6% and 73.5%, respectively, which were 9.2% and 11.1% higher than those of the corresponding alternatively modified sequences.

[0494] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si81-DV26 and P92-si81-DV27 were significantly improved by 17.7% and 15.5% compared to P92-si81-DV30, and by 15.8% and 13.6% compared to P92-si81-DV31, respectively.

[0495] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0496] After the basic sequence 81 was modified with templates DV21 and DV22, the inhibition rates of P92-si81-DV21 and P92-si81-DV22 on the PCSK9 gene were 78.3% and 75.1%, respectively, which were 15.9% and 12.7% higher than those of the alternatively modified sequences.

[0497] Using the modified templates DV26 and DV27 modified sequences disclosed herein, the inhibition rates of P92-si81-DV26 and P92-si81-DV27 were significantly improved by 11.0% and 8.8% compared with P92-si81-DV21, and by 14.2% and 12.0% compared with P92-si81-DV22, respectively.

[0498] From this we can see that:

[0499] 1) Base sequence 81, using the disclosed modified template DV25-29, significantly improved its inhibitory effect on the PCSK9 gene compared to the alternating modified sequence. For example, base sequence 81, using the DV26 modified sequence, showed a 26.9% higher inhibition rate than the alternating modified sequence.

[0500] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when the base sequence 81 was modified with the disclosed templates DV26 and DV27, the inhibition rate was significantly increased by up to 17.7% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 14.2%.

[0501] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0502] (4) Basic sequence 82

[0503] Table 17 PCSK9 gene inhibition rate after template modification of basic sequence 82

[0504] I. DV25-29 template of the present disclosure

[0505] Modification of base sequence 82 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 80%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV29 increased the inhibition rates of P92-si82-DV27 and P92-si82-DV29 by 19.6% and 19.4%, respectively.

[0506] II. Other modified templates disclosed herein

[0507] After the basic sequence 82 was modified with templates DV30 and DV31, the inhibition rates of P92-si82-DV30 and P92-si82-DV31 on the PCSK9 gene were 70.2% and 69.7%, respectively, which were comparable to the corresponding alternatively modified sequences.

[0508] Using the modified templates DV27 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si82-DV27 and P92-si82-DV29 were significantly improved by 19.1% and 18.9% compared to P92-si82-DV30, and by 19.6% and 19.4% compared to P92-si82-DV31, respectively.

[0509] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0510] After the basic sequence 82 was modified with templates DV21 and DV22, the inhibition rates of P92-si82-DV21 and P92-si82-DV22 on the PCSK9 gene were 76.5% and 75.4%, respectively, which were 6.8% and 5.7% higher than those of the alternatively modified sequences.

[0511] Using the modified templates DV27 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si82-DV27 and P92-si82-DV29 were significantly improved by 12.8% and 12.6% compared to P92-si82-DV21, and by 13.9% and 13.7% compared to P92-si82-DV22, respectively.

[0512] From this we can see that:

[0513] 1) Base sequence 82, using the disclosed modified template DV25-29, significantly improved PCSK9 gene inhibition compared to the alternating modified sequence. For example, base sequence 82, using the DV27 modified sequence, showed a 19.6% higher inhibition rate than the alternating modified sequence.

[0514] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when the base sequence 82 was modified with the disclosed templates DV27 and DV29, the inhibition rate was significantly increased by up to 19.6% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 13.9%.

[0515] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0516] (5) Basic sequence 84

[0517] Table 18 PCSK9 gene inhibition rate after template modification of basic sequence 84

[0518] I. DV25-29 template of the present disclosure

[0519] Modification of base sequence 84 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 85%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si84-DV28 and P92-si84-DV29 by 26.8% and 25.9%, respectively.

[0520] II. Other modified templates disclosed herein

[0521] After the basic sequence 84 was modified with templates DV30 and DV31, the inhibition rates of P92-si84-DV30 and P92-si84-DV31 on the PCSK9 gene were 70.3% and 72.4%, respectively, which were 8.7% and 10.8% higher than those of the corresponding alternatively modified sequences.

[0522] Using the modified templates DV28 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si84-DV28 and P92-si84-DV29 were significantly improved by 18.1% and 17.2% compared to P92-si84-DV30, and by 16.0% and 15.1% compared to P92-si84-DV31, respectively.

[0523] III. Prior Art Advanced ESC templates DV21 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, 11, 16, and 17) and DV22 (fluorinated sites: antisense strand positions 2, 6, 14, and 16, sense strand positions 7, 9, 10, and 11) have been disclosed.

[0524] After the basic sequence 84 was modified with templates DV21 and DV22, the inhibition rates of P92-si84-DV21 and P92-si84-DV22 on the PCSK9 gene were 77.7% and 75.1%, respectively, which were 16.1% and 13.5% higher than those of the alternatively modified sequences.

[0525] Using the modified templates DV28 and DV29 modified sequences disclosed herein, the inhibition rates of P92-si84-DV28 and P92-si84-DV29 were significantly improved by 10.7% and 9.8% compared to P92-si84-DV21, and by 13.3% and 12.4% compared to P92-si84-DV22, respectively.

[0526] From this we can see that:

[0527] 1) Base sequence 84, using the disclosed modified template DV25-29, significantly improved its inhibitory effect on the PCSK9 gene compared to the alternating modified sequence. For example, base sequence 84, using the DV28 modified sequence, showed a 26.8% increase in inhibition compared to the alternating modified sequence.

[0528] 2) The activity of the same siRNA sequence modified with different templates varied significantly. For example, when base sequence 84 was modified with the disclosed templates DV28 and DV29, the inhibition rate was significantly increased by up to 18.1% compared to when it was modified with the disclosed templates DV30 and DV31. Furthermore, when it was modified with the disclosed Advanced ESC templates DV21 and DV22, the inhibition rate was significantly increased by up to 13.3%.

[0529] 3) The activity of siRNA sequences modified with different templates varies greatly, up to 20%. Therefore, it is uncertain which template modification siRNA sequence will have the highest activity.

[0530] (6) Basic sequence 3

[0531] Table 19 PCSK9 gene inhibition rate after template modification of basic sequence 3

[0532] 1) Modification of base sequence 3 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si3-DV28 and P92-si3-DV29 by 24.0% and 24.7%, respectively.

[0533] 2) It can be seen that the basic sequence 3 using the modified template DV25-29 modified sequence disclosed herein has significantly improved the inhibitory activity on the PCSK9 gene compared to using the alternating modification.

[0534] (7) Basic sequence 8

[0535] Table 20 PCSK9 gene inhibition rate after template modification of basic sequence 8

[0536] 1) Modification of base sequence 8 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV28 increased the inhibition rates of P92-si8-DV27 and P92-si8-DV28 by 19.7% and 20.2%, respectively.

[0537] 2) It can be seen that the basic sequence 8 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0538] (8) Basic sequence 9

[0539] Table 21 PCSK9 gene inhibition rate after template modification of basic sequence 9

[0540] 1) Modification of base sequence 9 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV28 increased the inhibition rates of P92-si9-DV27 and P92-si9-DV28 by 27.2% and 25.4%, respectively.

[0541] 2) It can be seen that the basic sequence 9 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0542] (9) Basic sequence 21

[0543] Table 22 PCSK9 gene inhibition rate after template modification of basic sequence 21

[0544] 1) Modification of base sequence 21 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV25 and DV26 increased the inhibition rates of P92-si21-DV25 and P92-si21-DV26 by 24.4% and 26.5%, respectively.

[0545] 2) It can be seen that the base sequence 21 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0546] (10) Basic sequence 31

[0547] Table 23 PCSK9 gene inhibition rate after template modification of base sequence 31

[0548] 1) Modification of base sequence 31 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV25 and DV26 increased the inhibition rates of P92-si31-DV25 and P92-si31-DV26 by 22.9% and 22.6%, respectively.

[0549] 2) It can be seen that the base sequence 31 modified with the disclosed modification template DV25-29 has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0550] (11) Basic sequence 73

[0551] Table 24 PCSK9 gene inhibition rate after template modification of basic sequence 73

[0552] 1) Modification of base sequence 73 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 70%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV28 and DV29 increased the inhibition rates of P92-si73-DV28 and P92-si73-DV29 by 30.6% and 32.7%, respectively.

[0553] 2) It can be seen that the basic sequence 73 modified with the modified template DV25-29 disclosed herein has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0554] (12) Basic sequence 83

[0555] Table 25 PCSK9 gene inhibition rate after template modification of basic sequence 83

[0556] 1) Modification of base sequence 83 with the disclosed modification templates DV25-29 resulted in PCSK9 gene inhibition rates exceeding 75%. This significantly improved PCSK9 gene inhibition compared to alternating 2'-methoxy and 2'-fluoro modifications. For example, modification with templates DV27 and DV29 increased the inhibition rates of P92-si83-DV27 and P92-si83-DV29 by 30.9% and 29.7%, respectively.

[0557] 2) It can be seen that the basic sequence 83 modified with the modified template DV25-29 disclosed herein has a significantly improved inhibitory activity on the PCSK9 gene compared to the alternating modification.

[0558] Summarize:

[0559] (1) The selected base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, after modification with the modified templates DV25-29 designed in the present disclosure, showed significant inhibitory effects on PCSK9 gene expression, with inhibition rates 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.

[0560] (2) The 12 sequences described above, modified with the disclosed template DV25-29, showed significantly improved inhibition of PCSK9 gene expression compared to the alternatively modified sequences. For example, the inhibition of base sequence 51 modified with template DV27, P92-si51-DV27, was 29.1% higher than that of the alternatively modified sequence. The inhibition of base sequence 81 modified with template DV26, P92-si81-DV26, was 26.9% higher than that of the alternatively modified sequence.

[0561] (3) Modification of the same sequence with the disclosed modified templates DV25-29 significantly improved the inhibition rate of PCSK9 gene expression compared to modification templates disclosed in the prior art. For example, when base sequence 51 was modified with the disclosed modified template DV27, the inhibition rate increased by 21.3% compared to modification with the disclosed Advanced ESC template DV21.

[0562] (4) The same sequence modified with the disclosed modification templates DV25-29 showed significantly improved inhibition of PCSK9 gene expression compared to the modified templates DV30 and DV31. For example, the base sequence 51 modified with the disclosed modification template DV27 showed a 22.8% increase in inhibition compared to the modified template DV31.

[0563] (5) The activity of different template modification sequences varies greatly. For example, the inhibition rate of base sequence 5 modified with DV26 increased by 22.5% compared to that of base sequence 5 modified with DV31; the inhibition rate of base sequence 51 modified with DV27 increased by 20.5% compared to that of base sequence 51 modified with DV22. Therefore, it is uncertain which modification template can achieve high activity for siRNA sequences.

[0564] (ii) EC50 experiment

[0565] We selected a number of template modification sequences, 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, for a total of 12 sequences. EC50 experiments were performed to determine the EC50 concentration of each sequence. The experimental results are shown in Table 26.

[0566] Table 26 EC50 experimental results of each modified sequence

[0567] As can be seen from Table 26, 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. This indicates that these sequences can effectively inhibit PCSK9 gene expression at low concentrations.

[0568] Example 5: Comparison of the inhibitory effect of the sequence disclosed in the prior art on the PCSK9 gene

[0569] This example compares the inhibition efficiency of the PCSK9 gene by using the unmodified sequences disclosed in the prior art that are identical or similar to the basic sequences 3, 5, 8, 9, 31, 81, 82, 83 and 84 of the present disclosure, the alternatively modified sequences disclosed in the present disclosure, and the sequences modified using the DV25-29 template.

[0570] 1. Experimental Materials

[0571] Test samples:

[0572] (1) Prior art public sequence

[0573] Table 27 Prior Art Disclosed Sequences

[0574] Note: The sequences disclosed in the above patent applications are all unmodified sequence RNA.

[0575] (2) siRNA sequences modified with alternating 2'-OMe and 2'-F and terminal thiolation in Example 2: P92-si5, P92-si81, P92-si82, P92-si84, P92-si3, P92-si8, P92-si9, P92-si31, and P92-si83;

[0576] (3) The modified template DV25-29 was used to modify the siRNA sequence. The specific sequence is shown in Table 28.

[0577] Table 28 Sequences modified using DV25-29 modification template

[0578] Cell type: Hep3B cells, provided by Shanghai WuXi AppTec Co., Ltd. (ATCC-tings-1618164).

[0579] 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).

[0580] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0581] 2. Experimental Methods

[0582] qRT-PCR was used to detect the inhibitory effect of the test samples on the expression of PCSK9 gene mRNA in HEP3B cell line.

[0583] 2.1 Cell culture

[0584] Subcultured Hep3B cells were cultured in RPMI 1640 medium (100 μL / mL each of penicillin and streptomycin) with 10% fetal bovine serum in a 37°C, 5% CO2 incubator, with the medium changed daily. The cells were then digested and subcultured with 0.25% trypsin. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and fresh culture medium was added for subculture.

[0585] 2.2 Cell transfection

[0586] Add 2 μL of siRNA (1 μg / μL) to 18 μL of sterile, enzyme-free water at a 1:0.06 (wt / wt) ratio, mix thoroughly, and then add 22 μL of the prepared LNPs. Mix thoroughly and let stand at room temperature for 15 minutes. Aspirate 12.5 μL for encapsulation efficiency testing. The final transfection volume per well = (1 μg theoretical sample load / siRNA purity) / drug loading concentration, for a final siRNA concentration of 0.05 nM.

[0587] Negative control group: Add 22 μL of prepared LNPs to 20 μL of sterile enzyme-free water. Mix thoroughly and let stand at room temperature for 15 minutes.

[0588] After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured for 40 h.

[0589] 2.3 PCSK9 mRNA detection

[0590] 1) RNA extraction

[0591] The steps are the same as those in “1) RNA extraction” in “2.3 PCSK9 mRNA detection” in Example 2.

[0592] 2) RNA concentration detection

[0593] The steps are the same as those in "2) RNA concentration detection" in "2.3 PCSK9 mRNA detection" in Example 2.

[0594] 3) PCSK9 mRNA quantitative detection

[0595] In a 15 mL centrifuge tube, add the remaining components except primers and template in 7.5×48=360 portions, marked as A.

[0596] Label 1.5 mL EP tubes and add 77 μL of A7 and 420 ng of total RNA to each tube, mix well and set aside.

[0597] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 were mixed and set aside.

[0598] Add 1 μL of B1 and 1 μL of C to each well of the PCR plate. Cover with sealing film, centrifuge at 3000 rpm for 1 min, and transfer to the PCR machine.

[0599] *This step is performed on ice to maintain low temperature conditions.

[0600] Place the plate in the qPCR instrument and run the following program:

[0601] Reverse transcription: 55°C, 15 min;

[0602] Pre-denaturation: 95°C, 30 sec;

[0603] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0604] Melting curve: 95°C, 15 sec; 60°C, 60 sec; 95°C, 15 sec.

[0605] The run time was approximately 2 h. Analyze the experimental results and calculate 2-ΔΔCt.

[0606] 2.4 Data Processing

[0607] The calculation formula for PCSK9 mRNA expression rate (%) is:

[0608] Expression rate = (PCSK9 mRNA expression in the experimental group / PCSK9 mRNA expression in the blank control group) × 100%;

[0609] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0610] 3. Experimental Results

[0611] The specific experimental results are shown in Tables 29 and 30.

[0612] Experimental results demonstrate that the alternatively modified sequences disclosed herein and sequences modified with specific modification templates exhibit significantly enhanced PCSK9 inhibitory activity compared to identical or similar unmodified sequences disclosed in the prior art. For example, while the identical unmodified sequence si84 exhibited an inhibition rate of 38.6%, the alternatively modified sequence exhibited an inhibition rate of 61.6%, a 23.0% improvement over the unmodified sequence si84. After modification with the disclosed modification template DV26, the inhibition rate reached 86.8%, a 48.2% improvement over the unmodified sequence.

[0613] Compared with the sequence si31 disclosed in the prior art, the only difference between the sequence 31P disclosed in the present invention and the sequence si31 is that the 3' end of the antisense strand is missing two bases UG, and the rest are exactly the same. However, the inhibition rate of the unmodified sequence si31 disclosed in the present invention is 10.4% higher than that of 31P, and the inhibition rate of the alternatively modified sequence is 59.5%, which is 21.2% higher than that of 31P. After modification with the modified template DV26 disclosed in the present invention, the inhibition rate reached 80.2%, which is 41.9% higher than that of 31P.

[0614] (1) Compared with the identical unmodified sequences disclosed in the prior art, the alternating modified sequences disclosed in the present invention and the modified sequences using specific modification templates significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0615] Compared to identical sequences disclosed in the prior art, the alternatively modified sequences disclosed herein and sequences modified with specific modification templates significantly improved the inhibition rate of the PCSK9 gene. For example, the inhibition rate of the unmodified sequence si84 was 38.6%, while the inhibition rate after alternative modification was 61.6%, a 23.0% increase over the unmodified sequence si84. After modification with the disclosed modification template DV26, the inhibition rate reached 86.8%, a 48.2% increase over the unmodified sequence.

[0616] Table 29 Existing technology identical sequence inhibition rate of PCSK9 gene

[0617] The unmodified sequences si81, si84, si3, si8, si9, and si83 disclosed in the prior art in Table 29 are identical to the sequences disclosed herein. After modification with alternating modifications and the modified template disclosed herein, the inhibitory effect on PCSK9 was significantly improved. For example, the inhibition rate of the unmodified sequence si84 was 38.6%. After alternating modifications, the inhibition rate of P92-si84 was 61.6%, which was 23.0% higher than that of the unmodified sequence si84. After modification with the modified template DV25-29 disclosed herein, the inhibition rates of P92-si84-DV25, P92-si84-DV26, P92-si84-DV27, P92-si84-DV28, and P92-si84-DV29 reached 84.9%, 86.8%, 84.5%, 86.2%, and 86.4%, respectively, all of which were more than 40% higher than those of the unmodified sequence si84.

[0618] (2) Compared with the unmodified sequences disclosed in the prior art, which have only minor differences, the unmodified sequences, the alternatively modified sequences, and the sequences modified with specific modification templates disclosed in the present invention significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0619] Compared to sequences disclosed in the prior art with only minor differences, the unmodified sequences, alternatively modified sequences, and sequences modified with specific modification templates disclosed herein significantly improved the inhibition rate of the PCSK9 gene. For example, the unmodified sequence 31P had an inhibition rate of 38.3%, while the similar unmodified sequence si31 disclosed herein had an inhibition rate of 48.7%, a 10.4% increase over 31P. The alternatively modified sequence had an inhibition rate of 59.5%, a 21.2% increase over 31P. After modification with the disclosed modification template DV26, the inhibition rate reached 80.2%, a 41.9% increase over 31P.

[0620] Table 30: PCSK9 gene inhibition rate of similar sequences in the prior art and the present disclosure

[0621] I. Sequence Alignment

[0622] The sequences of the prior art disclosed sequences 5P, 31P and 82P in Table 30 are very close to the sequences si5, si31 and si82 disclosed in the present invention, with only minor differences. Specific comparisons are shown in the table below:

[0623] Table 31 Comparison of sequences disclosed in the prior art and sequences disclosed herein

[0624] As can be seen from Table 31, the sequence 5P disclosed in the prior art is only missing two bases GG at the 3' end of the antisense chain compared with the sequence si5 disclosed in the present invention; the sequence 31P is only missing two bases UG at the 3' end of the antisense chain compared with the sequence si31 disclosed in the present invention; the sequence 82P is only missing two bases AA at the 3' end of the antisense chain compared with the sequence si82 disclosed in the present invention, and the other sequences are exactly the same.

[0625] II. Activity Comparison

[0626] 1) The unmodified sequences disclosed in this paper showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 disclosed in this paper showed a 10.4% higher inhibition rate than the structurally similar 31P.

[0627] The unmodified sequence si5 disclosed in the present invention has an inhibition rate of 50.1% on the PCSK9 gene, which is 8.3% higher than the similar sequence 5P disclosed in the prior art (inhibition rate of 41.8%), a significant improvement.

[0628] The unmodified sequence si82 disclosed in the present invention has an inhibition rate of 53.8% on the PCSK9 gene, which is 7.1% higher than the similar sequence 82P disclosed in the prior art (inhibition rate of 46.7%), a significant improvement.

[0629] The unmodified sequence si31 disclosed in the present invention has an inhibition rate of 48.7% on the PCSK9 gene, which is 10.4% higher than the similar sequence 31P disclosed in the prior art (inhibition rate of 38.3%), a significant improvement.

[0630] 2) The alternatively modified sequences disclosed in this disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 5P, the inhibition rate of the alternatively modified sequence P92-si5 disclosed in this disclosure increased by 22.0%.

[0631] After the basic sequence 5 disclosed in the present invention is alternately modified, the inhibition rate of P92-si5 on the PCSK9 gene is 63.8%, which is 22.0% higher than that of the unmodified sequence 5P disclosed in the prior art, a significant improvement.

[0632] After the basic sequence 82 disclosed in the present invention is alternately modified, the inhibition rate of P92-si82 on the PCSK9 gene is 68.3%, which is 21.6% higher than that of the unmodified sequence 82P disclosed in the prior art, a significant improvement.

[0633] After the basic sequence 31 disclosed in the present invention is alternately modified, the inhibition rate of P92-si31 on the PCSK9 gene is 59.5%, which is 21.2% higher than that of the unmodified sequence 31P disclosed in the prior art, a significant improvement.

[0634] 3) Sequences modified with the disclosed modified templates showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 82P, the disclosed alternatively modified sequence P92-si82-DV29 showed a 42.8% higher inhibition rate.

[0635] After modification of the disclosed basic sequence 5 with modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 5P disclosed in the prior art. For example, P92-si5-DV27 increased by 40.8%, a significant improvement.

[0636] After modification of the disclosed base sequence 82 with the modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 82P disclosed in the prior art. For example, P92-si82-DV29 increased the inhibition rate by 42.8%, a significant improvement.

[0637] After modification of the base sequence 31 with the modified templates 25-29, the inhibition rate was increased by more than 30% compared to the unmodified sequence 31P disclosed in the prior art. For example, P92-si31-DV26 increased by 41.9%, a significant improvement.

[0638] It can be seen that compared with similar unmodified sequences in the prior art, the unmodified sequences disclosed herein, and the alternatingly modified and template-modified sequences disclosed herein can significantly enhance the inhibitory activity against PCSK9, with the inhibition rate being increased by up to 40%.

[0639] Summarize:

[0640] (1) Compared with identical sequences disclosed in the prior art, the alternatively modified sequences and sequences modified with specific modification templates disclosed herein significantly enhance the inhibitory effect on the PCSK9 gene. For example, the inhibition rate of the unmodified sequence si84 was 38.6%, while the inhibition rate after alternative modification was 61.6%, a 23.0% increase over the unmodified sequence si84. After modification with the modified template DV26 disclosed herein, the inhibition rate reached 86.8%, a 48.2% increase over the unmodified sequence.

[0641] (2) Compared with similar sequences disclosed in the prior art, the unmodified sequences, alternatively modified sequences, and sequences modified using a specific modification template disclosed in the present invention have significantly improved inhibitory effects on the PCSK9 gene. For example, the unmodified sequence si31 disclosed in the present invention has an inhibition rate increased by 10.4% compared with the 31P sequence disclosed in the prior art, which is similar to the sequence disclosed in the prior art. The sequence si5 disclosed in the present invention is alternatively modified to the sequence P92-si5, and the inhibition rate is increased by 22.0% compared with the unmodified sequence 5P disclosed in the prior art, which is similar to the sequence si5. The sequence si82 disclosed in the present invention is modified using the modified template DV29 disclosed in the present invention, the sequence P92-si82-DV29, and the inhibition rate is increased by 42.8% compared with the unmodified sequence 82P disclosed in the prior art, which is similar to the sequence si82.

[0642] Example 6: Inhibitory effect of PCSK9 and off-target genes using sequences designed to prevent off-target effects

[0643] 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 siRNAs is primarily due to off-target effects, resulting in gene suppression of the wrong target through a recognition mechanism similar to that of microRNA (miRNA).

[0644] To address this issue, Alnylam's latest fifth-generation template design incorporates a glycol nucleic acid (GNA) modification at position 7 of the siRNA antisense strand to disrupt the antisense strand's seed region, significantly mitigating off-target effects and alleviating hepatotoxicity. This modification can influence siRNA binding to non-targets through recognition in the seed region, thereby suppressing off-target effects.

[0645] Natural or simple direct 5' phosphorylation may lead to dephosphorylation within cells. Direct 5' phosphorylated oligonucleotides can be 90% dephosphorylated after 2 hours of circulation in the blood, with complete dephosphorylation occurring after 24 hours. The 5'-phosphorylated design (5'-E-VP) utilizes E-vinylphosphonate in place of the bridging oxygen, resulting in optimal phosphorylation and higher stability.

[0646] In summary, in order to reduce the off-target effect of the sequence and examine the effect of 5' end phosphorylation on off-target efficacy, this example uses the basic sequences 5, 51, 81 and 84 modified with DV25-29, and adds 5'-E-VP modification and 7-position GNA anti-off-target modification at the 5' end of the antisense strand.

[0647] In addition, the sequences P92-si3, P92-si8, P92-si21, P92-si31, P92-si73 and P92-si83 modified with alternating 2'-methoxy and 2'-fluoro groups, and the above sequences P92-si3+, P92-si8+, P92-si21+, P92-si31+, P92-si73+ and P92-si83+ containing the antisense chain 7-position GNA anti-off-target modification design were compared to detect off-target effects.

[0648] The experimental results showed that at a concentration of 10 nM, the sequences using the anti-off-target design had a significant inhibitory effect on the target gene PCSK9, while the inhibitory effect on off-target genes was significantly reduced. This indicates that the anti-off-target design does not affect the inhibitory effect of the disclosed alternating modified and template modified sequences on the PCSK9 gene, but can significantly suppress off-target effects.

[0649] 1. Experimental Materials

[0650] 1) Test sample:

[0651] Alternating modified sequences: Alternating modified sequences P92-si3, P92-si8, P92-si9, P92-si21, P92-si31, P92-si73 and P92-si83 were used.

[0652] Sequences of alternating modifications and anti-off-target design: P92-si3+, P92-si8+, P92-si9+, P92-si21+, P92-si31+, P92-si73+, and P92-si83+ (Table 32).

[0653] The basic sequences 5, 84, 81 and 51 used template modified sequences, sequences using template modification and 5'-E-VP modification, sequences using template modification and anti-off-target design, and sequences using template modification, 5'-E-VP modification and anti-off-target design (Table 33).

[0654] 2) Sequence synthesis:

[0655] I. Alternating Modified Sequences and Template Modified Sequences

[0656] The synthesis method is as in Example 1.

[0657] II. siRNA sequences containing 5'-E-VP

[0658] Referring to Example 1, the siRNA sequence was synthesized. When synthesizing the base at the 5' end of the antisense strand (the last base), a monomer containing a phosphonic acid group at the 5' end was used, for example, a vinyl-(E)-phosphonate-A-OMe phosphoramidite monomer (Formula 9) or a vinyl-(E)-phosphonate-U-OMe phosphoramidite monomer (Formula 10). Examples of their structures are as follows:

[0659] III. siRNA sequences with anti-off-target design

[0660] The siRNA sequence was synthesized with reference to Example 1. When synthesizing the antisense strand from the 5' end to the 7th base, a GNA monomer was used to synthesize the sequence. The structure of the GNA monomer is as follows:

[0661] Table 32 Alternative modification plus anti-off-target modification sequence

[0662] The above sequences all adopt the alternating modification scheme of 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) at the 2' position.

[0663] (1) All odd-numbered positions of the sense strand are modified with 2'-F, and all even-numbered positions are modified with 2'-OMe;

[0664] (2) The odd-numbered positions of the antisense strand are all modified with 2'-OMe, and the even-numbered positions are all modified with 2'-F;

[0665] (3) There are two thiolation modifications at the 5' end of the sense strand and at both the 5' and 3' ends of the antisense strand;

[0666] (4) The 7th base of the antisense strand (5'-3' direction) was modified with GNA.

[0667] Table 33 Template modification, 5'-E-VP modification, and anti-off-target design sequences

[0668] Cell type: Hep3B cells, provided by Shanghai WuXi AppTec Co., Ltd. (ATCC-tings-1618164).

[0669] 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).

[0670] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0671] 2. Experimental Methods

[0672] 1) Compound dilution

[0673] For off-target experiments, the test samples were diluted to a concentration of 10 nM and tested in triplicate wells.

[0674] Hep3B cell digestion and counting:

[0675] Hep3B cells were taken out when the density reached 80%, rinsed with Dulbecco's phosphate buffered saline (DPBS), and digested with 0.05% trypsin for 3-10 min. After the cells were collected, they were counted using Countstar Rigel S2 and the number of cells was adjusted to 2×10 5 / mL density.

[0676] 2) Preparation of transfection reagent

[0677] Prepare RNAiMAX transfection reagent at a ratio of 3:97 RNAiMAX:Opti-MEM in a 15 mL centrifuge tube. Vortex for 15 seconds to mix thoroughly, and incubate at room temperature for 15 minutes. Add 40 μL of RNAiMAX Opti-MEM to each well of the corresponding position. Add 40 μL of the diluted compound of the corresponding concentration to the corresponding wells of the dilution plate, mix thoroughly, and incubate for 15 minutes.

[0678] 3) Cell plating

[0679] Hep3B cells (2×10 4 cells / well) to a 96-well cell plate, and the siRNA compound was mixed with RNAiMAX Opti-MEM transfection reagent and then added to the cells in each well. At the same time, a control group containing RNAiMAX Opti-MEM without siRNA compound was set up.

[0680] Off-target prevention assay: Add the compound and RNAiMAX Opti-MEM mixture from the dilution plate to a 96-well cell culture plate (20 μL / well). Subsequently, add 100 μL / well of cells to the 96-well plate for a final volume of 120 μL per well. After plating, incubate in a 5% CO2, 37°C incubator for 24 h.

[0681] 4) RNA extraction and reverse transcription

[0682] 24 hours after transfection, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using QIAGEN 96 Kit (QIAGEN-74182), and cDNA was synthesized using FastKing RT Kit (with gDNase) (TIANGEN-KR116-02) according to the manufacturer's instructions.

[0683] 5) RT-qPCR

[0684] Target cDNA was detected by qPCR, and GAPDH cDNA was detected in parallel as an internal control. 8 μL of the prepared qPCR reaction solution and 2 μL of sample cDNA were added to 384-well plates. The TaqMan qPCR reaction program was as follows: heating at 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles. The SYBR qPCR reaction program was as follows: heating at 50°C for 2 minutes, heating at 95°C for 10 minutes, then cycling at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles; the final melting curve was performed by heating at 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds.

[0685] 6) Data Analysis

[0686] The target gene RNA expression level in each sample was calculated based on the Ct value using the ΔΔCt relative quantification method. The relative expression of the target gene was expressed as 2-ΔΔCT.

[0687] The calculation formula is as follows:

[0688] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0689] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group);

[0690] Relative expression of target gene mRNA = 2-ΔΔCT

[0691] Inhibition rate = (1-relative expression level of sample / average expression level of RNAiMAX control) × 100%

[0692] 3. Experimental Results

[0693] (1) Different modified sequences all had significant inhibitory effects on the PCSK9 gene. For example, the inhibition rate of the alternating modification P92-si3 reached 75.6%, and the inhibition rate of the template modification and 5'-E-VP modification sequence D84-DV27P was as high as 94.4%.

[0694] The inhibition rates of PCSK9 gene by all tested samples are shown in Tables 34-36.

[0695] All the alternatively modified sequences had significant inhibitory effects on PCSK9 at a concentration of 10 nM. For example, the inhibition rate of the alternatively modified sequence P92-si3 reached 75.6%.

[0696] I. The alternating modified sequences have a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[0697] Table 34 Inhibition rate of alternating modified sequence PCSK9 gene

[0698] As can be seen from the above table, at a concentration of 10 nM, the alternatively 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 them, P92-si3 had the highest inhibition rate, reaching 75.6%.

[0699] II. Template modification sequences employing off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P achieved an inhibition rate of up to 94.4%.

[0700] Table 35 Template modification and inhibition rate of PCSK9 gene using anti-off-target or / and 5'-E-VP modified sequence

[0701] 1) After modification with the disclosed modification templates, base sequences 5, 51, 81, and 84 exhibited significant inhibition against the PCSK9 gene, with inhibition rates exceeding 90%. Furthermore, the inhibitory effect was further enhanced compared to alternatively modified sequences. For example, after alternatively modifying base sequence 51, the inhibition rate of P92-si51 was 70.1% (see Table 34). After modification with template DV26, the inhibition rate of D51-DV26 reached 90.7%, a significant 20.6% increase compared to the alternatively modified sequences (Figure 8).

[0702] 2) After template modification and anti-off-target design, the researchers demonstrated significant inhibitory activity against PCSK9 gene expression. For example, when the base sequence 84 was modified with the template DV26 and then subjected to anti-off-target design, the inhibition rate of D84-DV26+ reached 90.1%.

[0703] 3) Template modification followed by 5'-E-VP modification can enhance the inhibitory effect on the PCSK9 gene. For example, when base sequence 84 was modified with template DV27, the inhibition rate of D84-DV27 was 91.5%. After 5'-E-VP modification, the inhibition rate of D84-DV27P was 94.4%, an increase of 2.9%.

[0704] 4) After template modification, followed by off-target protection and 5'-E-VP modification, inhibitory activity against the PCSK9 gene can reach over 90%. For example, base sequence 84 was modified with template DV27, followed by off-target protection and 5'-E-VP modification, and the inhibition rate of D84-DV27+P reached 90.4%.

[0705] This demonstrates that using the disclosed modified template DV25-29 to modify a sequence can further enhance the inhibitory effect compared to alternatively modified sequences. Furthermore, sequences modified with the disclosed modified template DV25-29, followed by off-target design and / or 5'-E-VP modification, significantly inhibit PCSK9 gene expression, with inhibition rates exceeding 90%.

[0706] (2) After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[0707] The experimental results show that:

[0708] 1) The alternating modification or template modification sequence disclosed herein, with an anti-off-target design, can reduce the inhibitory effect on off-target genes, i.e., has a significant anti-off-target effect;

[0709] 2) By using the template-modified sequence, anti-off-target design and 5'-E-VP modification, the inhibition rate of off-target genes was significantly reduced, indicating a significant anti-off-target effect.

[0710] I. The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0711] Table 36 Inhibition rate of off-target genes by alternating modification and its anti-off-target modification sequence

[0712] As can be seen from Table 36, after the alternate modified sequences of the present disclosure adopt an anti-off-target design, the inhibition rate of off-target genes is reduced, showing an anti-off-target effect. For example, the alternate modified sequence P92-si3+ adopting an anti-off-target design has a 20.0% lower inhibition rate for the off-target gene AARSD1 and a 20.5% lower inhibition rate for the off-target gene ACAP2 than the sequence P92-si3 without an anti-off-target design, which is a significant reduction.

[0713] II. Using the disclosed modification template to modify the sequence using an anti-off-target design has a significant anti-off-target effect, and the inhibition rate of off-target genes can be reduced by up to 73.6%.

[0714] Table 37 Template modification sequence and its anti-off-target and / or 5'-E-VP modification inhibition rate on off-target genes

[0715] 1) Only use the anti-off-target design (indicated by "+" in the sequence number)

[0716] After the template-modified sequence was designed to prevent off-target effects, the inhibitory effect on off-target genes was significantly reduced. For example, the inhibition rate of D84-DV26+ on the MXD1 gene was reduced by 39.9%, the inhibition rate of D81-DV25+ on the PCYOX1 gene was reduced by 54.1%, the inhibition rate of D51-DV26+ on the NEPRO gene was reduced by 25.9%, and the inhibition rate of D5-DV25+ on the DTWD1 gene was reduced by 10.9%.

[0717] It shows that after the basic sequences 5, 51, 81 and 84 are modified with the modification template designed in the present disclosure and subjected to the anti-off-target design, they all have significant anti-off-target effects.

[0718] 2) Simultaneous use of off-target protection and 5'-E-VP modification (indicated by "+P" in the sequence numbering)

[0719] The use of both template-modified and 5'-E-VP-modified sequences significantly reduced the inhibition rate of off-target genes, demonstrating a significant anti-off-target effect. For example, D84-DV26+P reduced the inhibition rate of the off-target gene MXD1 by 73.6%, D81-DV27+P reduced the inhibition rate of the off-target gene PCYOX1 by 31.1%, and D51-DV26+P reduced the inhibition rate of the off-target gene KIF1B by 41.6%, significantly reducing the inhibition rate.

[0720] It shows that after the basic sequences 5, 51, 81 and 84 are modified with the modification template designed by the present disclosure, and the anti-off-target design and 5'-E-VP modification are simultaneously adopted, there is a significant off-target effect.

[0721] Summarize:

[0722] 1. All modified sequences showed significant inhibitory effects on the PCSK9 gene. For example, the alternating modification P92-si3 achieved an inhibition rate of 75.6%, while the template-modified and 5'-E-VP modified sequence D84-DV27P achieved an inhibition rate of 94.4%.

[0723] (1) The alternating modified sequences had a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[0724] (2) Template modification sequences using an anti-off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P has an inhibition rate of up to 94.4%.

[0725] 2. After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[0726] (1) The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0727] (2) The modified sequence of the modified template disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by up to 73.6%.

[0728] Example 7: Effects of sequences modified with the disclosed modified template on PCSK9, low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) in mouse serum

[0729] In this example, several sequences, including base sequences 5, 51, 81, 82, and 84, were modified using, for example, template modification alone, template modification combined with an anti-off-target design, template modification combined with a 5'-E-VP design, and template modification, 5'-E-VP, and an anti-off-target design. Transgenic mice expressing the human PCSK9 gene were used to test the inhibitory effects of these sequences on serum PCSK9, low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) at different time points using ELISA.

[0730] 1. Experimental Materials

[0731] Test drug:

[0732] The sequences in Table 38 include sequences using only template modification, sequences using template modification and anti-off-target design, sequences using template modification and anti-off-target design, and sequences using template modification, 5'-E-VP and anti-off-target design simultaneously. The 3' end of the sense strand of these sequences is coupled to the GalNAc ligand G4, G5, G6 or G7:

[0733] The method for coupling the oligonucleotide to the ligand G4, G5, G6, or G7 is the same as the method for preparing conjugates 4, 5, 6, and 7 in Example 3 of patent application CN116854754A, namely, coupling YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 to the oligonucleotide. The synthesis method of YK-GAL-304, YK-GAL-305, YK-GAL-306, and YK-GAL-307 is the same as that described in Example 1 of the same patent application.

[0734] The oligonucleotide forms a conjugate with the ligand as shown below:

[0735] The specific sequences of each sequence are shown in Table 38. G4, G5, G6 and G7 in the sequence number indicate that the sequence is coupled with the GalNAc ligand G4, G5, G6 or G7.

[0736] Table 38 Sequences in animal experiments

[0737] Preparation of test drug:

[0738] Drug solvent: PBS buffer

[0739] Preparation conditions: sterile environment

[0740] Labeling method: The prepared drug preparation shall be labeled with the special number, name, concentration, quantity, preparation date, preparer and storage conditions on the outer packaging;

[0741] Storage conditions: Prepare immediately before use and store remaining samples at -80℃.

[0742] Experimental animal information:

[0743] Species / Strain: hPCSK9 transgenic mice

[0744] Rating: SPF

[0745] Gender: Male

[0746] Quantity: 214

[0747] Age: 4 to 7 weeks

[0748] Weight: 18~20g

[0749] Source: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0750] Production license number: SCXK(Su)2018-0008

[0751] Laboratory Animal Ethics Review (IACUC):

[0752] The experimental animals were housed at Youji (Tianjin) Pharmaceutical Technology Co., Ltd., license number: SYXK (Jinbin) 2019-0002. This project has been reviewed by the Experimental Animal Ethics Committee of Youji (Tianjin) Pharmaceutical Technology Co., Ltd., and the experimental procedures were conducted in strict accordance with IACUC requirements to ensure animal welfare.

[0753] Feeding and management:

[0754] Animal husbandry: Experimental animals were housed at Youji (Tianjin) Pharmaceutical Technology Co., Ltd. (license number: SYXK (Jinbin) 2019-0002). Cages were maintained with dimensions of 29.0 cm (length × width × height) × 18.5 cm × 13.0 cm. The temperature was set between 20 and 26°C, the humidity between 40% and 70%, and the air was exchanged at least 15 times per hour with fresh air. Artificial lighting was maintained with a 12-hour light and 12-hour dark cycle.

[0755] The breeding environment conditions are based on the National Standard of the People's Republic of China GB14925-2010, and the environment is controlled by a modular air-conditioning unit.

[0756] Animals were allowed to eat and drink freely. The drinking water bottles and the drinking water in the bottles should be changed at least twice a week. After use, the drinking water bottles should be sterilized in a pulse vacuum sterilizer before reuse.

[0757] Animal cages and bedding should be changed at least once a week. All animal cages and bedding should be sterilized under high pressure in a pulsating vacuum sterilizer before being used in a barrier environment. Animal cages should be cleaned, disinfected and wiped at least once a week.

[0758] The animal breeding and observation room is cleaned and disinfected every day, including the flat racks, floors, tables, etc.

[0759] The disinfectants used in the barrier environment include: 6.67% chlorhexidine solution, 0.5% 84 disinfectant, 75% disinfectant, and 0.08% Baidusai. The four disinfectants should be used in turn and cannot be mixed.

[0760] Experimental animal feed: SPF rat maintenance feed: produced by Sibeifu (Beijing) Biotechnology Co., Ltd., animal feed production license number is SCXK (Beijing) 2019-0010, issued by Beijing Municipal Science and Technology Commission.

[0761] Feed Testing: Each batch of feed is certified for quality. Microbiological testing is conducted quarterly by our company, and every six months, the feed supplier provides a recent third-party feed testing report. Feed nutrient content testing is conducted in accordance with the National Standard of the People's Republic of China GB14924.3-2010, while contaminant index testing is conducted in accordance with the National Standard of the People's Republic of China GB14924.2-2001.

[0762] Drinking water for experimental animals: Drinking water: sterile water prepared by the filtration system, directly filled in drinking bottles.

[0763] Drinking Water Testing: We conduct microbiological testing quarterly and send water to a third-party testing agency for annual water quality testing. Drinking water testing is conducted in accordance with the National Standard of the People's Republic of China, GB5749-2006.

[0764] Animal bedding: Corn cob bedding: Produced by Sibefor (Beijing) Biotechnology Co., Ltd., animal bedding production license number is SCXK(Beijing)2019-0004, issued by Beijing Municipal Science and Technology Commission.

[0765] Litter Testing: We conduct microbiological testing quarterly, and the bedding supplier provides at least one third-party bedding testing report every six months. Bedding testing is conducted in accordance with the National Standard of the People's Republic of China, GB14924.2-2001.

[0766] 2. Experimental Methods

[0767] Dosage design and grouping

[0768] Definition of experimental day: The day on which the animals were administered with the vehicle or the test drug was defined as day 0.

[0769] Grouping and Dosing: After the experimental animals were acclimated to feeding, they were randomly divided into a negative control group and a test drug group based on serum PSCK9 protein levels, with 5 animals in each group. The drug was administered via a single subcutaneous injection at a dose of 6 mg / kg in a volume of 1 mL / kg at a concentration of 6 mg / mL. The day of administration was designated as day 0.

[0770] Animals are individually identified using ear tags. Cages are identified by hanging cage labels. A laboratory sign is hung at the laboratory entrance. Grouping information is detailed in the table below:

[0771] Table 39

[0772] Detection indicators

[0773] (1) General observation

[0774] The subjects were observed once a day from one week before administration to the end of the experiment.

[0775] Observation content: Observe the animal's death or dying, mental state, behavioral activities, fecal characteristics, feed and water supply, etc. beside the cage.

[0776] Test animals: all animals in the negative control group and the test drug group.

[0777] (2) Expression of PCSK9 protein in serum

[0778] Testing time: before dosing on day 3 (day-3), 1 week after dosing on day 7 (1w), 2 weeks after dosing on day 14 (2w), 3 weeks after dosing on day 21 (3w), 4 weeks after dosing on day 28 (4w), and 5 weeks after dosing on day 35 (5w).

[0779] PCSK9 protein level detection method: ELISA kit is used for detection; serum samples should be kept away from repeated freezing and thawing.

[0780] Test animals: all animals in the negative control group and the test drug group.

[0781] (3) Blood lipid measurement

[0782] Testing time: Before dosing on day 3 (day 3), one week after dosing on day 7 (1 week), two weeks after dosing on day 14 (2 weeks), three weeks after dosing on day 21 (3 weeks), four weeks after dosing on day 28 (4 weeks), and five weeks after dosing on day 35 (5 weeks). Approximately 200 μL of blood was collected from the inner canthus of the eye. The whole blood sample was temporarily stored in an ice box before centrifugation. Centrifuge at 2-8°C and a centrifugal force of approximately 3000 g for 10 minutes. The sample was aliquoted into two tubes (one aliquot of approximately 60 μL, with the remaining serum in the other tube) and stored at -70-86°C for lipid testing.

[0783] (4) Data processing and statistical analysis

[0784] The experimental data are expressed as mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 8.3 software. Homogeneity of variance was tested using the LSD test, while unequal variance was tested using the Dunnett T3 test. P < 0.05 was considered statistically significant.

[0785] 3. Experimental Results

[0786] Specific experimental results are shown in Tables 40-42.

[0787] It can be seen that these sequences can significantly and continuously inhibit PCSK9 protein in serum and significantly reduce serum low-density lipoprotein cholesterol (LDL-C) and serum total cholesterol (TC) levels. For example, the D81-DV25G7 group achieved an inhibition rate of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively; the D82-DV27PG5 group reduced serum LDL-C levels by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively; and the D82-DV29PG7 group reduced serum TC levels by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[0788] (1) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, exhibited significant inhibitory effects on PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively.

[0789] Table 40 Different sequences inhibit PCSK9 protein in serum

[0790] As can be seen from Table 40, the conjugates formed by conjugating each sequence with a GalNAc compound have a significant inhibitory effect on PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively ( FIG9 ).

[0791] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the animal liver and significantly inhibit PCSK9 gene expression.

[0792] (2) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, can significantly reduce serum low-density lipoprotein cholesterol (LDL-C) levels. For example, the D82-DV27PG5 group achieved reductions of 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively.

[0793] Table 41 Effects of different sequences on the reduction of serum low-density lipoprotein cholesterol (LDL-C)

[0794] As can be seen from Table 41, serum LDL-C levels were significantly reduced in all experimental groups. For example, in the D82-DV27PG5 group, serum LDL-C levels were reduced by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively ( FIG10 ).

[0795] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the liver of animals and significantly reduce the level of low-density lipoprotein cholesterol (LDL-C) in serum.

[0796] (3) Sequences disclosed herein, such as base sequences 5, 51, 81, 82, and 84, with various modifications, can significantly reduce serum total cholesterol (TC) levels. For example, D82-DV29PG7 reduced total cholesterol by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

[0797] Table 42 Different sequences reduce serum total cholesterol (TC) levels

[0798] As can be seen from Table 42, serum total cholesterol (TC) was significantly reduced in all experimental groups. For example, in the D82-DV29PG7 group, TC was reduced by 32.2%, 26.8% and 19.6% on days 7, 14 and 28, respectively ( FIG11 ).

[0799] It shows that the basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified with the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with GalNAc compounds, can be efficiently delivered to the liver of animals and significantly reduce the total cholesterol (TC) level in serum.

[0800] Summarize:

[0801] The basic sequences designed in the present invention, such as 5, 51, 81, 82 and 84, are modified using the template of the present invention, or simultaneously modified with 5'-E-VP and / or anti-off-target design, and conjugated with a GalNAc compound. They can be efficiently delivered to the liver of animals and can significantly and sustainably inhibit PCSK9 protein expression in serum, thereby reducing serum low-density lipoprotein cholesterol (LDL-C) and serum total cholesterol (TC) levels.

[0802] For example, the inhibition rate of PCSK9 protein expression in the serum of the D81-DV25G7 group reached 83.1%, 82.2% and 70.3% on the 7th, 14th and 28th days, respectively; the serum low-density lipoprotein cholesterol (LDL-C) level of the D82-DV27PG5 group decreased by 32.6%, 35.8% and 21.7% on the 7th, 14th and 28th days, respectively; the serum total cholesterol (TC) level of the D82-DV29PG7 group decreased by 32.2%, 26.8% and 19.6% on the 7th, 14th and 28th days, respectively.

[0803] in conclusion:

[0804] This paper designs a series of siRNAs based on the PCSK9 mRNA sequence, modifies them alternately and with a set of specific modification templates, and also performs off-target design on some of these sequences with 5'-E-VP modification. The results show that:

[0805] (1) Unmodified sequences, including basic sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, all have significant inhibitory effects on PCSK9, with the highest inhibition rate exceeding 60%.

[0806] (2) Using multiple modified sequences, the inhibition rate can reach up to 90%. Moreover, the modified sequence is conjugated with a GalNAc compound and can be efficiently delivered to the animal liver, significantly inhibiting PCSK9 gene expression, significantly reducing serum low-density lipoprotein cholesterol (LDL-C) levels, and significantly reducing serum total cholesterol (TC) levels.

[0807] The details are as follows:

[0808] 1. The disclosed alternative modified sequence has a significant inhibitory effect on the PCSK9 gene

[0809] (1) Among the 84 designed sequences, 12 sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, 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. Among them, the inhibition rates of P92-si5, P92-si81, P92-si82, P92-si3, P92-si8, and P92-si31 exceeded 70%.

[0810] (2) The inhibition rates of the 26 sequences on the PCSK9 gene ranged from 30% to 50%. For example, the inhibition rates of P92-si6 and P92-si7 were 38.3% and 35.7%, respectively.

[0811] (3) The inhibition rates of 46 sequences on the PCSK9 gene were below 30%. For example, the inhibition rates of P92-si1 and P92-si20 were only 9.2% and 6.0%, respectively.

[0812] (4) siRNAs with similar sequences have very different activities. For example, the inhibition rate of P92-si5 is 61.8% higher than that of P92-si4, which is a significant improvement.

[0813] 2. The unmodified sequence disclosed herein has a significant inhibitory effect on the PCSK9 gene

[0814] (1) 12 unmodified sequences showed significant inhibitory effects on the PCSK9 gene, with inhibition rates exceeding 50%, including si5, si51, si81, si82, si84, si3, si8, si9, si21, si31, si73, and si83. Among them, si5, si81, si82, si3, and si8 showed inhibition rates exceeding 60%.

[0815] (2) The inhibition rates of other unmodified sequences on the PCSK9 gene were all lower than 40%. For example, the inhibition rates of si52 and si74 were only 0.5% and 2.2% respectively.

[0816] (3) siRNAs with similar sequences have very different activities. For example, the inhibition rate of base sequence 5 is 51.2% higher than that of base sequence 4, which is a significant improvement.

[0817] (4) The effects of alternating modifications on the activity of different sequences were not consistent. Some showed significant increases in inhibition rate, such as the base sequence 5, where the inhibition rate of alternating modifications was 12.4% higher than that of the unmodified sequence. Others showed little difference, such as the base sequences 4, 22, and 52, where the inhibition rates of the alternating modified sequences and the unmodified sequences remained essentially unchanged.

[0818] 3. The sequence modified by the modified template disclosed herein has a significant inhibitory effect on the PCSK9 gene

[0819] (1) Base sequences 5, 51, 81, 82, 84, 3, 8, 9, 21, 31, 73, and 83, after modification with the modified templates DV25-29 designed in this disclosure, showed significant inhibitory effects on PCSK9 gene expression, with inhibition rates exceeding 70%. Among them, P92-si81-DV26, P92-si82-DV27, and P92-si82-DV29 achieved inhibition rates of 89.3%, 89.3%, and 89.1%, respectively.

[0820] (2) The 12 sequences described above, modified with the disclosed template DV25-29, showed significantly improved inhibition of PCSK9 gene expression compared to the alternatively modified sequences. For example, the inhibition of base sequence 51 modified with template DV27, P92-si51-DV27, was 29.1% higher than that of the alternatively modified sequence. The inhibition of base sequence 81 modified with template DV26, P92-si81-DV26, was 26.9% higher than that of the alternatively modified sequence.

[0821] (3) Modification of the same sequence with the disclosed modified templates DV25-29 significantly improved the inhibition rate of PCSK9 gene expression compared to modification templates disclosed in the prior art. For example, when base sequence 51 was modified with the disclosed modified template DV27, the inhibition rate increased by 21.3% compared to modification with the disclosed Advanced ESC template DV21.

[0822] (4) The same sequence modified with the disclosed modification templates DV25-29 showed significantly improved inhibition of PCSK9 gene expression compared to the modified templates DV30 and DV31. For example, the base sequence 51 modified with the disclosed modification template DV27 showed a 22.8% increase in inhibition compared to the modified template DV31.

[0823] (5) The EC50 values ​​of the above 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. This indicates that these sequences can effectively inhibit PCSK9 gene expression at low concentrations.

[0824] 4. The sequences modified by alternating modification and modification template disclosed in the present invention have significantly improved PCSK9 inhibitory activity compared with the unmodified sequences disclosed in the prior art that are completely identical or have very little difference.

[0825] (1) Compared with the identical unmodified sequences disclosed in the prior art, the alternating modified sequences disclosed in the present invention and the modified sequences using specific modification templates significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0826] (2) Compared with the unmodified sequences disclosed in the prior art, which have only minor differences, the unmodified sequences, the alternatively modified sequences, and the sequences modified with specific modification templates disclosed in the present invention significantly improve the inhibition rate of the PCSK9 gene, which can be increased by up to 40%.

[0827] I. The unmodified sequences disclosed in this disclosure have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, the unmodified sequence si31 disclosed in this disclosure has a 10.4% higher inhibition rate than the structurally similar 31P.

[0828] II. The alternatively modified sequences disclosed herein have significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 5P, the inhibition rate of the alternatively modified sequence P92-si5 disclosed herein was increased by 22.0%.

[0829] III. Sequences modified with the disclosed modified templates showed significantly improved activity compared to similar unmodified sequences disclosed in the prior art. For example, compared to sequence 82P, the disclosed alternatively modified sequence P92-si82-DV29 showed a 42.8% higher inhibition rate.

[0830] 5. The alternating modified sequence and template modified sequence disclosed herein adopt a specific anti-off-target design, which has a significant inhibitory effect on the PCSK9 gene and significantly reduces the inhibitory effect on off-target genes.

[0831] (1) Different modified sequences all had significant inhibitory effects on the PCSK9 gene. For example, the inhibition rate of the alternating modification P92-si3 reached 75.6%, and the inhibition rate of the template modification and 5'-E-VP modification sequence D84-DV27P was as high as 94.4%.

[0832] I. The alternating modified sequences have a significant inhibitory effect on the PCSK9 gene, with inhibition rates exceeding 60%, with P92-si3 reaching 75.6%.

[0833] II. Template modification sequences employing off-target design and / or 5'-E-VP modification have a significant inhibitory effect on the PCSK9 gene. For example, the template modification and 5'-E-VP modification sequence D84-DV27P achieved an inhibition rate of up to 94.4%.

[0834] (2) After adopting any one or more of the various modification methods disclosed in the present invention, anti-off-target design is performed, which has a significant anti-off-target effect on off-target genes and reduces the inhibitory effect on off-target genes.

[0835] I. The alternating modified sequence disclosed herein adopts an anti-off-target design, which has a significant anti-off-target effect and can reduce the inhibition rate of off-target genes by 20%.

[0836] II. Using the disclosed modification template to modify the sequence using an anti-off-target design has a significant anti-off-target effect, and the inhibition rate of off-target genes can be reduced by up to 73.6%.

[0837] 6. The modified sequences using the disclosed modified templates significantly inhibited PCSK9 expression in mouse serum and significantly reduced low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels.

[0838] (1) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly inhibited PCSK9 protein expression in serum. For example, D81-DV25G7 achieved inhibition rates of 83.1%, 82.2%, and 70.3% on days 7, 14, and 28, respectively.

[0839] (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, the D82-DV27PG5 group reduced LDL-C by 32.6%, 35.8%, and 21.7% on days 7, 14, and 28, respectively.

[0840] (3) Different modifications of the disclosed base sequences 5, 51, 81, 82, and 84 significantly reduced serum total cholesterol (TC). For example, D82-DV29PG7 reduced TC by 32.2%, 26.8%, and 19.6% on days 7, 14, and 28, respectively.

Claims

1. A double-stranded RNAi agent for reducing the expression of PCSK9, comprising any oligonucleotide duplex selected from the following sense and antisense strand pairs: (1) The sense strand has a sequence as shown in SEQ ID NO. 1 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 13 or a fragment thereof, or a modified sequence thereof; (2) the sense strand has a sequence as shown in SEQ ID NO. 2 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 14 or a fragment thereof, or a modified sequence thereof; (3) the sense strand has a sequence as shown in SEQ ID NO. 3 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 15 or a fragment thereof, or a modified sequence thereof; (4) the sense strand has a sequence as shown in SEQ ID NO. 4 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 16 or a fragment thereof, or a modified sequence thereof; (5) the sense strand has the sequence shown in SEQ ID NO. 5 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 17 or a fragment thereof, or a modified sequence thereof; (6) the sense strand has a sequence as shown in SEQ ID NO. 6 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 18 or a fragment thereof, or a modified sequence thereof; (7) the sense strand has a sequence as shown in SEQ ID NO. 7 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 19 or a fragment thereof, or a modified sequence thereof; (8) The sense strand has the sequence shown in SEQ ID NO. 8 or a fragment thereof, or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO. 20 or a fragment thereof, or a modified sequence thereof; (9) the sense strand has a sequence as shown in SEQ ID NO. 9 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 21 or a fragment thereof, or a modified sequence thereof; (10) The sense strand has a sequence as shown in SEQ ID NO. 10 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 22 or a fragment thereof, or a modified sequence thereof; (11) the sense strand has a sequence as shown in SEQ ID NO. 11 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 23 or a fragment thereof, or a modified sequence thereof; (12) The sense strand has a sequence as shown in SEQ ID NO. 12 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO. 24 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

2. The double-stranded RNAi agent according to claim 1, wherein all nucleotides on the sense strand and the antisense strand are modified nucleotides. The double-stranded RNAi agent according to claim 1 or 2, which is an RNAi agent for inhibiting PCSK9 gene expression. The double-stranded RNAi agent according to claim 1 or 2, wherein the sense strand differs from any one of SEQ ID NOs. 1-12 by 1-3 nucleotides. The double-stranded RNAi agent according to claim 1 or 2, wherein the antisense strand differs from any one of SEQ ID NOs. 13-24 by 1-3 nucleotides.

6. The double-stranded RNAi agent according to claim 1 or 2, wherein at least one modified nucleotide is selected from the group consisting of: deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

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

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

9. The double-stranded RNAi agent according to claim 1 or 2, wherein the double-stranded region is 15-30 nucleotide pairs in length.

10. The double-stranded RNAi agent according to claim 1 or 2, wherein the double-stranded region is 17-25 nucleotide pairs in length. The double-stranded RNAi agent according to claim 1 or 2, wherein the double-stranded region is 19-23 nucleotide pairs in length.

12. The double-stranded RNAi agent according to claim 1 or 2, wherein the double-stranded region is 21 nucleotide pairs in length.

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

14. The double-stranded RNAi agent of claim 1 or 2, wherein each strand has 19-25 nucleotides. The double-stranded RNAi agent according to claim 1 or 2, wherein the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides. The double-stranded RNAi agent according to claim 1 or 2, wherein all nucleotide modifications on the sense strand and the antisense strand are chemical modifications of the 2' position of the ribose sugar of the nucleotide.

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

20. The double-stranded RNAi agent according to claim 1 or 2, wherein the chemical modification of the 2' position of each nucleotide ribose is as follows: the odd-numbered positions of the sense strand are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified; the odd-numbered positions of the antisense strand are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified. The double-stranded RNAi agent according to claim 1 or 2, wherein the nucleotide monomers are connected by 3', 5'-phosphodiester bonds. The double-stranded RNAi agent according to claim 1 or 2, wherein the 3',5'-phosphodiester bonds connecting the nucleotide monomers are thio-modified.

23. The double-stranded RNAi agent according to claim 1 or 2, further comprising the following modifications: The antisense strand is modified in one of the following ways: The sense strand is modified in one of the following ways: In the above table, 2'-OMe is 2'-methoxy; 2'-F is 2'-fluoro; PS is a phosphorothioate backbone; The antisense strand adopts modification A, and the sense strand adopts modification a; The antisense strand adopts modification B, and the sense strand adopts modification a; The antisense strand adopts modification C, and the sense strand adopts modification a; The antisense strand adopts modification B, and the sense strand adopts modification b; The antisense strand adopts modification C, and the sense strand adopts modification b.

24. The double-stranded RNAi agent according to claim 1 or 2, wherein the antisense strand adopts a modification group at the second to eighth positions from the 5' end, wherein the modification group is selected from UNA, GNA or DNA, wherein the structures of UNA and GNA are as follows: in, The base is selected from adenine, guanine, cytosine, thymine and uracil.

25. The double-stranded RNAi agent according to claim 1 or 2, wherein the phosphorylation of the 5'-carbon atom of the 5'-terminal nucleotide glycoside of the modified antisense strand includes, but is not limited to, the following 5'-phosphorylation groups: 5'-vinylphosphonate group (5'-E-VP); 5'-methylphosphonate group (5'-MP); 5'-C-methylphosphonate group; 5'-thiophosphate group (5'-PS); 5'-phosphate group (5'-P), the structure of which is shown below: in, R is hydrogen, hydroxyl, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy, C 1-4 alkylcarbonylamino or halogen; The base is selected from adenine, guanine, cytosine, thymine and uracil.

26. The double-stranded RNAi agent according to claim 1 or 2, wherein the 3',5'-phosphodiester bonds connecting the nucleotide monomers at the ends of the sequence are thio-modified to form chirally pure 3',5'-phosphothioate diester bonds, wherein the 5' ends of the sense strand and the antisense strand contain 1-3 thio linkages, and the 3' end of the antisense strand contains 1-3 thio linkages.

27. A conjugate for reducing the expression of PCSK9, comprising the double-stranded RNAi agent according to any one of claims 1 to 26, and a ligand conjugated thereto. The conjugate according to claim 27 , wherein the ligand is conjugated to the 3′-end or 5′-end of the sense strand of the oligonucleotide.

29. The conjugate of claim 27 or 28, wherein the ligand is one or more GalNAc derivatives attached using a divalent or trivalent branched linkage.

30. The conjugate of claim 27 or 28, wherein the ligand is: in, X is hydrogen or a hydroxyl protecting group or H, wherein the hydroxyl protecting group includes acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer between 0 and 20; q, r and s are independently integers between 1 and 7.

31. The conjugate of claim 30, wherein the ligand is:

32. The conjugate of claim 27 or 28, wherein the ligand is: in, X is oxygen, nitrogen or sulfur; Y is an alkyl group or an aromatic group; R1 is oxygen or sulfur; R2 is hydrogen, amino, C 1-4 Alkyl, aromatic, C 1-4 Alkoxy or 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 -, where e is an integer from 0 to 7; L is -CONH- or -NHCO-; X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5; X2 is -(CH2) g -, 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.

33. The conjugate according to claim 32, wherein the ligand is G4, G5, G6 or G7:

34. The conjugate according to claim 27 or 28, which has the structure shown below:

35. The conjugate of claim 27 or 28, wherein the double-stranded RNAi agent comprises any one oligonucleotide duplex selected from the following sense and antisense strand pairs: (1) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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) 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; and (16) 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; in, The oligonucleotide duplex is conjugated to ligand G4, G5, G6 or G7.

36. The conjugate of claim 27 or 28, wherein the double-stranded RNAi agent comprises any one oligonucleotide duplex selected from the following sense and antisense strand pairs: (1) 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) 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; and (3) 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; in, The oligonucleotide duplex is conjugated to ligand G4, G5, G6 or G7.

37. The conjugate according to claim 27 or 28, wherein the double-stranded RNAi agent comprises an oligonucleotide duplex consisting of a sense strand represented by SEQ ID NO. 353 and an antisense strand represented by SEQ ID NO. 448, and the oligonucleotide duplex is conjugated to ligand G5.

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

39. Use of the double-stranded RNAi agent of any one of claims 1-26, the conjugate of any one of claims 27-37, or the composition of claim 38 in the preparation of a method for treating a disease associated with PCSK9.

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

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