siRNA targeting PCSK9 gene and its application

Abstract

The present invention provides an oligonucleotide or a modified sequence thereof for treating diseases related to PCSK9. 80 small interfering RNA (siRNA) sequences were designed for the PCSK9 mRNA sequence and were modified alternately. Cell experiment results show that some unmodified or modified siRNA sequences have a significant inhibitory effect on PCSK9 gene expression and can be used to develop drugs for treating and / or preventing diseases related to PCSK9 gene expression.

Description

Technical Field

[0001] The present disclosure relates to an oligonucleotide, in particular to a small interfering ribonucleic acid (siRNA) for treating and / or preventing diseases related to the expression of PCSK9 gene. Background Art

[0002] Nucleic acid drugs, especially oligonucleotide drugs, are widely used due to their simple synthesis and high activity. Oligonucleotide drugs generally include antisense oligonucleotides (ASO), small interfering RNA (siRNA), micro RNA (miRNA) and nucleic acid aptamers.

[0003] Oligonucleotides are a class of short DNA or RNA molecules, oligomers, that readily bind to their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes, or less commonly, higher order hybrids. This fundamental property makes oligonucleotides widely used in genetic testing, targeted gene therapy research, and medicine. These small fragments of nucleic acid can be manufactured as single-stranded molecules with any specified sequence. In nature, oligonucleotides are usually small RNA molecules that play a role in the regulation of gene expression, or degradation intermediates derived from the decomposition of larger nucleic acid molecules.

[0004] RNA interference is a natural defense mechanism against foreign genes. siRNA can knock out target genes by recognizing specific sequences and degrading target mRNA.

[0005] The effective molecules of classical RNA interference are composed of the iconic 19+2 nucleotide polymer structure, that is, a double helix structure composed of 21 nucleotide RNA molecules and 19 corresponding base nucleotide molecules, including a 2-nucleotide 3' end overhang. One strand of siRNA, the guide strand or antisense strand, is complementary to the mRNA transcript of the target gene, and the other strand is labeled as the passenger strand or sense strand. The siRNA antisense strand guides Arguin to complement the target transcript and become part of the RNA-guided silencing complex. The complete complementarity of the siRNA antisense strand with the target leads to the breakage of the target transcript at the opposite position of the antisense strand 10-11 under the catalysis of Arguin.

[0006] siRNA has a natural advantage over small molecule therapies and antibody drugs because siRNA performs its function by completing Watson–Crick base pairing with mRNA, while small molecule and monoclonal antibody drugs need to recognize the complex spatial structure of specific proteins. Therefore, many diseases cannot be treated by small molecules and monoclonal antibodies because their target molecules are highly active and it is difficult to identify and design specific structures with affinity. In contrast, genes of interest can be targeted by siRNA, and the mechanism of action of siRNA gives it a shorter research and development cycle and a wider range of treatments than small molecule or antibody drugs.

[0007] Antisense oligonucleotides connected by phosphodiester backbones can bind to complementary target RNA chains through their sequence specificity and can induce ribonuclease H to cleave target RNA after hybridization. However, under physiological conditions, phosphodiester bonds are particularly sensitive to nucleases, so natural, unmodified, and unmodified oligonucleotide drugs are easily and rapidly degraded by nucleases in vivo, resulting in low activity and poor drugability. Since antisense oligonucleotides have poor stability and are easily degraded by nucleases in vivo, chemical modification of synthetic antisense oligonucleotides can increase the serum stability of antisense oligonucleotides, thereby effectively inhibiting the expression of target genes.

[0008] Chemical modification of oligonucleotide structure is an effective way to improve its activity. Chemically modified and transformed oligonucleotides can improve their stability to nucleases, affinity to RNA, and better promote cellular endocytosis and tissue targeting, thereby improving their activity. 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 and transformation technology of nucleic acid drugs has been continuously upgraded.

[0009] Based on the basic structure of oligonucleotides, namely the base, sugar ring, phosphate backbone and end, chemical modification can be performed in four parts.

[0010] Base modification: mainly divided into three forms: purine modification, pyrimidine modification and base substitution. Purine modification includes N6-methyladenosine, N1-methyladenosine, 7-methylguanylate modification; pyrimidine modification includes 3-methyluridine, 5-methyluridine, 5-methylcytosine, N4-acetylcytidine, pseudouridine, thiouridine, propyne uridine and dihydrouridine.

[0011] Sugar ring modification: mainly divided into sugar ring modification and replacement. Sugar ring modification includes 2'-modification, 4'-modification, 5'-modification and isomeric modification, as well as the combination of these modifications.

[0012] Modification of the phosphate backbone: Since the hydrolysis of the phosphodiester bond of oligonucleotides by nucleases in the body is the main reason for the rapid degradation of oligonucleotides in the body, choosing other suitable analogs to replace the phosphodiester bond backbone can increase its stability. The main modification methods of the phosphate backbone are: thiophosphate, methyl phosphate, selenophosphate, methyl borophosphate or dithiophosphate; or replacing the entire phosphate group between nucleosides with a group that does not contain a phosphorus atom, such as replacing the P atom with C, S and N atoms to form a guanidine group, S-methylthiourea, etc.

[0013] Modification of the ends: Covalent coupling of special groups to the 5' or / and 3' ends of siRNA can enhance affinity with cells, tissue selectivity and cell targeting. Modification of fat-soluble small molecules at the ends can improve their compatibility with cell membranes and increase the efficiency of cell entry. The asialoglycoprotein receptor (ASGPR) is an endocytic receptor specifically expressed by hepatocytes. The coupling of ASGPR's high-affinity ligand N-acetylgalactosamine (GalNAc) with siRNA can achieve liver-targeted delivery of oligonucleotide drugs. In 2019, Alnynam's siRNA drug (Givlaari) modified with GalNAc technology was approved by the FDA for the treatment of acute hepatic porphyria.

[0014] Thanks to the progress of the above modification technology, the development of nucleic acid drugs has been rapidly developed. So far, 16 nucleic acid drugs have been approved for marketing worldwide, as shown in Table 1:

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

[0016]

[0017] Hypercholesterolemia refers to elevated serum total cholesterol or low-density cholesterol levels, which is a risk factor for diseases such as atherosclerosis, coronary heart disease and stroke. The current treatment strategy for hypercholesterolemia is mainly to reduce LDL-C (low-density lipoprotein cholesterol) that causes atherosclerosis and increase HDL-C (high-density lipoprotein cholesterol) that has a potential cardioprotective effect. Clinical studies from nearly 170,000 subjects have shown that for every 1.0 mmol / L reduction in LDL-C levels, the average annual incidence of major cardiovascular disease events can be reduced by about 20%.

[0018] PCSK9 is a serine protease encoded by the PCSK9 gene, which is mainly produced by the liver. PCSK9 binds to the LDL receptors on the surface of liver cells and degrades the LDL receptors. The LDL receptors have the function of binding to LDL-C in plasma and absorbing it into the liver (processing the blood lipids therein and excreting them through bile). The degradation of LDL receptors will lead to an increase in the LDL-C level in plasma. PCSK9 inhibitors increase the level of LDL receptors on the surface of liver cells by reducing the expression level of PCSK9, thereby reducing the LDL-C level in plasma and achieving the purpose of lowering blood lipids.

[0019] Currently, targeted PCSK9 inhibitors are available on the market, including antibodies and small nucleic acid drugs, for the treatment of hypercholesterolemia and related diseases. However, other inhibitors targeting this target are still being developed in this field in the hope of achieving better therapeutic effects or higher safety. Summary of the invention

[0020] In this regard, the present disclosure designs 80 small interfering RNA (siRNA) sequences for PCSK9 mRNA sequence, including 40 sense strands and 40 antisense strands, and performs 2'-F and 2'-Me alternating modifications on these sequences.

[0021] The designed unmodified and modified oligonucleotide sequences were tested for their inhibitory activity on PCSK9 expression. The results showed that the above oligonucleotide sequences were able to significantly reduce the expression of the PCSK9 gene in in vitro cell experiments, indicating that they can be used to develop drugs to treat related diseases caused by high cholesterol.

[0022] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, wherein the oligonucleotide comprises any oligonucleotide duplex selected from the following sense strand and antisense strand pairing:

[0023] (1) the sense strand has a sequence as shown in SEQ ID NO.40 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.80 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0024] (2) the sense strand has a sequence as shown in SEQ ID NO.32 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.72 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0025] (3) the sense strand has a sequence as shown in SEQ ID NO.6 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.46 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0026] (4) the sense strand has a sequence as shown in SEQ ID NO.20 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.60 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0027] (5) the sense strand has a sequence as shown in SEQ ID NO.33 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.73 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0028] (6) the sense strand has a sequence as shown in SEQ ID NO.35 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.75 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0029] (7) the sense strand has a sequence as shown in SEQ ID NO.14 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.54 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0030] (8) the sense strand has a sequence as shown in SEQ ID NO.29 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.69 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0031] (9) the sense strand has a sequence as shown in SEQ ID NO.31 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.71 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0032] (10) The sense strand has a sequence as shown in SEQ ID NO.38 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.78 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0033] In another aspect, the present disclosure provides use of an oligonucleotide or a pharmaceutically acceptable salt or conjugate or composition thereof in the preparation of a medicament for treating and / or preventing diseases associated with PCSK9 gene expression, including diseases related to lipid disorders.

[0034] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned conjugate, and optionally a pharmaceutically acceptable carrier.

[0035] In another aspect, the present disclosure provides a use of an oligonucleotide or a pharmaceutically acceptable salt or conjugate or composition thereof in the preparation of a drug for treating or / and preventing diseases related to the expression of the PCSK9 gene, including diseases related to lipid disorders. The beneficial effects achieved by the present invention are at least as follows:

[0036] 1) Some of the 80 unmodified siRNA sequences designed, including 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 paired double-stranded siRNA, had a significant inhibitory effect on the expression of PCSK9 mRNA. For example, the inhibition rate of the 35 / 75 group reached 68.90%, and the inhibition rates of the 6 / 46, 32 / 72 and 40 / 80 groups also reached more than 60%.

[0037] 2) The designed 80 oligonucleotide sequences were modified with 2'-F and 2'-Me alternately. The activity test results showed that the double-stranded siRNA paired with the alternately modified sequences 88 / 128, 114 / 154 and 122 / 162 had an inhibition rate of more than 70% on PCSK9 gene expression.

[0038] 3) The inventors found that the 80 designed siRNA sequences were modified alternately with 2'-F and 2'-Me. This modification method had different effects on the activity of different sequences. Some of them could significantly increase their inhibition rate, up to 36.8%; some of them significantly reduced the inhibition rate, up to 17.2%; and some of them had no change in activity after modification.

[0039] 4) The inventors also found that not all siRNA sequences designed for PCSK9 mRNA sequences (including unmodified or 2'-F and 2'-Me alternating modified sequences) have inhibitory activity on PCSK9 mRNA expression, and different sequences can have very large differences. It takes a lot of creative work to screen out oligonucleotide sequences that have significant inhibitory effects on PCSK9 mRNA. DETAILED DESCRIPTION

[0040] In order to make the present disclosure more easily understood, certain 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 the present disclosure.

[0041] As used herein, the articles "a" and "an" refer to one or more than one (ie, 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.

[0042] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to."

[0043] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0044] 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 specified or in addition apparent from context, the term "approximately" or "approximately" refers to 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 numeral will exceed 100% of possible value).

[0045] As used herein, "PCSK9" refers to the proprotein convertase subtilisin Kexin9 gene or protein.

[0046] "G", "C", "A" and "U" each generally represent nucleotides comprising guanine, cytosine, adenine and uracil as bases, respectively. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleotides 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 will be well aware that guanine, cytosine, adenine and uracil may be replaced by other moieties without substantially changing 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 may base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide comprising uracil, guanine or adenine may be replaced in the nucleotide sequences of the present disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such replacement moieties are embodiments of the present disclosure.

[0047] The terms "RNAi agent", "iRNA", "iRNA agent", "RNA interfering agent" are used interchangeably herein to refer to RNA agents as defined herein and to mediate targeted cleavage of RNA transcripts through the RNA induced silencing complex (RISC) pathway. RNAi directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). RNAi modulates, e.g., inhibits, 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).

[0048] The term "small interfering RNA" or "siRNA" refers to a small interfering RNA RNAi molecule. It is a class of double-stranded RNA molecules, also referred to in the art as short interfering RNA or silencing RNA. siRNAs typically comprise a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a leading strand), wherein each strand is 17 to 30 nucleotides in length, typically 19 to 25 nucleosides in length, wherein the antisense strand is complementary to the target nucleic acid (such as at least 95% complementary, such as fully complementary) (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 strand may form a blunt-ended duplex, or preferably, the 3' ends of the sense and antisense strands may form a 3' overhang, such as 1, 2 or 3 nucleosides, similar to the product produced by Dicer, which may form a RISC substrate 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 2nt 3' overhangs. Thus, the length of the duplex region can be, for example, 17 to 25 nucleotides, such as 21 to 23 nucleotides in length.

[0049] 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 "complementarity region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) as defined herein. When the complementary region is not completely complementary to the target sequence, the mismatch may be in the interior or terminal regions of the molecule. Typically, the most tolerated mismatch is in the terminal region, e.g., within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

[0050] The term "sense strand" as used herein refers to the strand of a RNAi that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0051] As used herein, the term "inhibit" may be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0052] 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) and a variant (such as a naturally occurring variant) or mutant of a 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.

[0053] "Inhibiting PCSK9 gene expression" includes any level of inhibition of the PCSK9 gene, such as at least partial inhibition of the expression of the PCSK9 gene, 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%.

[0054] 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 controlled subject, cell, or sample (e.g., only a buffer control or an inert agent control) treated subject.

[0055] 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.

[0056] As used herein, "PCSK9-related diseases" are 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 PCSK9 gene mutations, by abnormal cleavage of PCSK9 protein, by abnormal interactions between PCSK9 and other proteins or other endogenous or exogenous substances. Exemplary PCSK9-related diseases include lipidemias, such as hyperlipidemia, and other forms of lipid imbalances, such as hypercholesterolemia, hypertriglyceridemia, and pathological conditions associated with these disorders, such as heart and circulatory system diseases.

[0057] As used herein, "therapeutically effective amount" is intended to include an amount of RNAi agent sufficient to achieve treatment of a PCSK9-related disease (e.g., by attenuating, ameliorating, or maintaining the existing disease or one or more disease symptoms) when administered to a patient. 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.

[0058] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or improve a disease or one or more symptoms of a PCSK9-related disease when administered to a subject who has not yet experienced or shown 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 degree of risk 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.

[0059] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces a certain desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0060] 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, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs or local areas. For example, a sample may be derived from a fluid or cell in a specific organ, an organ part or these organs. In certain embodiments, a sample may be derived from liver (for example, some segments of the whole liver or liver, or some types of cells in the liver, for example, hepatocytes). In a preferred embodiment, "sample derived from a subject" refers to blood or plasma extracted from the subject. In other embodiments, "sample derived from a subject" refers to liver tissue (or its subcomponent) derived from the subject.

[0061] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, wherein the oligonucleotide comprises any oligonucleotide duplex selected from the following sense strand and antisense strand pairing:

[0062] (1) the sense strand has a sequence as shown in SEQ ID NO.40 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.80 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0063] (2) the sense strand has a sequence as shown in SEQ ID NO.32 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.72 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0064] (3) the sense strand has a sequence as shown in SEQ ID NO.6 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.46 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0065] (4) the sense strand has a sequence as shown in SEQ ID NO.20 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.60 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0066] (5) the sense strand has a sequence as shown in SEQ ID NO.33 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.73 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0067] (6) the sense strand has a sequence as shown in SEQ ID NO.35 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.75 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0068] (7) the sense strand has a sequence as shown in SEQ ID NO.14 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.54 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0069] (8) the sense strand has a sequence as shown in SEQ ID NO.29 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.69 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0070] (9) the sense strand has a sequence as shown in SEQ ID NO.31 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.71 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0071] (10) The sense strand has a sequence as shown in SEQ ID NO.38 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.78 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0072] In some embodiments, the sense strand is 19-25 nucleotides in length.

[0073] In some embodiments, the antisense strand is 19-27 nucleotides in length.

[0074] In some embodiments, the modified oligonucleotide comprises a 2'-modification.

[0075] In some embodiments, the 2'-modification is selected from the group consisting of 2'-O-methyl, 2'-O-methoxyethyl, 2'-fluoro, 2'-O-benzyl, and 2'-O-methyl-4-pyridine.

[0076] In some embodiments, the nucleotide monomers in the oligonucleotide sequence are linked by internucleotide bonds, preferably, the internucleotide bonds are 3',5'-phosphodiester bonds.

[0077] In some embodiments, the oligonucleotide comprises any one oligonucleotide duplex selected from the following sense and antisense strand pairs:

[0078] (1) the sense strand has a sequence as shown in SEQ ID NO.122 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.162 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0079] (2) the sense strand has a sequence as shown in SEQ ID NO.114 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.154 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0080] (3) the sense strand has a sequence as shown in SEQ ID NO.88 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.128 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0081] (4) the sense strand has a sequence as shown in SEQ ID NO.102 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.142 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0082] (5) the sense strand has a sequence as shown in SEQ ID NO.115 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.155 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0083] (6) the sense strand has a sequence as shown in SEQ ID NO.117 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.157 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0084] (7) the sense strand has a sequence as shown in SEQ ID NO.96 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.136 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0085] (8) the sense strand has a sequence as shown in SEQ ID NO.111 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.151 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0086] (9) the sense strand has a sequence as shown in SEQ ID NO.113 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.153 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof;

[0087] (10) The sense strand has a sequence as shown in SEQ ID NO.120 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.160 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0088] In another aspect, the present disclosure provides a conjugate for reducing the expression of PCSK9, comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and a targeting ligand conjugated thereto.

[0089] In some embodiments, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0090] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0091] In some embodiments, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0092] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof or the aforementioned conjugate, and optionally a pharmaceutically acceptable carrier.

[0093] In some embodiments, the lipid disorder is hyperlipidemia, atherosclerosis and / or one or more symptoms or complications thereof; more preferably, the hyperlipidemia is hypercholesterolemia;

[0094] The other drugs for treating hyperlipidemia are selected from fibrates, statins, bile acid sequestrants and nicotinic acids.

[0095] In another aspect, the present disclosure provides use of an oligonucleotide or a pharmaceutically acceptable salt or conjugate or composition thereof in the preparation of a medicament for treating and / or preventing diseases associated with PCSK9 gene expression, including diseases related to lipid disorders.

[0096] In some embodiments, the lipid disorder is hyperlipidemia, atherosclerosis, and / or one or more symptoms or complications thereof.

[0097] In some embodiments, the hyperlipidemia is hypercholesterolemia.

[0098] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. 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.

[0099] The present disclosure designs 80 small interfering RNA (siRNA) sequences for PCSK9 mRNA sequence, including 40 sense strands and 40 antisense strands, and adds TsT modification (s is thio) to the 3' end of each strand to increase the stability of the sequence, and these sequences are modified with 2'-F and 2'-Me alternately (the odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand are all 2'-F modified, and the even-numbered positions of the sense strand and the odd-numbered positions of the antisense strand are all 2'-OMe modified). The positive control of the unmodified oligonucleotide sequence is PC (Inclisiran drug sequence of Alnylam Company), and the 3' end of each strand of the positive control is modified with TsT. The unmodified oligonucleotide sequence is shown in Table 2, and the oligonucleotides modified with 2'-F and 2'-Me alternately are shown in Table 3.

[0100] Table 2 Unmodified oligonucleotide sequences

[0101]

[0102]

[0103] Table 3 Modified oligonucleotides

[0104]

[0105]

[0106] Example 1: Synthesis of oligonucleotides

[0107] The unmodified oligonucleotide sequences SEQ ID NO.1-80 and positive controls SEQ ID NO.81 and SEQ ID NO.82 in Table 2, the modified oligonucleotide sequences SEQ ID NO.83-162 in Table 3, and the modified oligonucleotide sequences SEQ ID NO.163 and SEQ ID NO.164 obtained by the same alternating modification method of the positive controls SEQ ID NO.81 and SEQ ID NO.82 were synthesized by chemical methods.

[0108] 1. Synthesis of Modified Oligonucleotides

[0109] 1.1 Synthesis of modified oligonucleotide sequence 88 in Table 3

[0110] The unmodified oligonucleotide sequence corresponding to the modified oligonucleotide sequence 88 in Table 3 is SEQ ID NO.6:

[0111] 5'-CACCAGAUCCUGCAUGUCUUCCTsT-3'(SEQ ID NO.6)

[0112] SEQ ID NO. 6 is a positive strand, and the odd-numbered positions are all modified with 2'-fluoro (2'-F), and the even-numbered positions are all modified with 2'-methoxy (2'-OMe).

[0113] Instruments and reagents: OligoPilot 400 synthesizer from GE, USA, with a solid phase carrier of cross-linked polystyrene beads, model Primer support 5GUnylinker350 (manufactured by Cytiva).

[0114] Preparation method:

[0115] According to the monomer concentration of 0.15M, the following nucleotide monomer solutions were prepared with acetonitrile:

[0116] 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), DMT-UF phosphoramidite monomer (Formula 8) and DMT-dT phosphoramidite monomer (Formula 9).

[0117]

[0118]

[0119] Prepared by the following steps:

[0120] 1) Deprotection

[0121] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotection agent, followed by washing with acetonitrile.

[0122] 2) Coupling

[0123] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolyl as an activating agent, followed by washing with acetonitrile.

[0124] (3) Oxidation / sulfurization

[0125] Oxidation: Oxidation was carried out using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by washing with acetonitrile.

[0126] Sulfurization: A 3% pyridine solution of hydrogenated xanthan gum was used as a sulfurizing agent for sulfurization, followed by washing with acetonitrile.

[0127] 4) Hydroxyl protection

[0128] 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 washing with acetonitrile.

[0129] Repeat the above steps and cycle according to the set sequence to obtain a fully protected product.

[0130] 5) Deprotection

[0131] A 3% dichloroacetic acid toluene solution was used as a deprotection agent to remove the DMT protecting group of the last nucleotide, followed by washing with acetonitrile.

[0132] 6) Ammonolysis and purification

[0133] The solid phase carrier is transferred to a reactor, concentrated ammonia water (25-28%) is added, and after maintaining the aminolysis at 60°C for 12 hours, the system is cooled to room temperature and the mixture is transferred to a filter press, 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.

[0134] 7) Annealing

[0135] The purified sense strand and antisense strand were mixed in a 1:1 ratio, heated to 95°C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double strand.

[0136] 1.2 Synthesis of other modified sequences in Table 3

[0137] According to the above synthesis method, other modified oligonucleotides in Table 3 were synthesized, wherein the odd-numbered sites of the sense strand and the even-numbered sites of the antisense strand were modified with 2'-F, and the other sites were modified with 2'-OMe.

[0138] 2. Synthesis of unmodified oligonucleotides

[0139] The nucleotide monomers are DMT-A (bz) phosphoramidite monomer, DMT-C (bz) phosphoramidite monomer, DMT-G (ibu) phosphoramidite monomer and DMT-U phosphoramidite monomer. The above-mentioned synthesis method is used to synthesize 80 unmodified oligonucleotide sequences and unmodified positive control sequences in Table 2.

[0140] Example 2: Inhibitory effect of unmodified oligonucleotides on the PCSK9 gene

[0141] The unmodified oligonucleotide sequences 1-80 synthesized in Example 1 and the positive controls 81-82 were tested for their inhibitory activity on the PCSK9 gene. The results showed that the double-stranded siRNA paired with the unmodified sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 had a significant inhibitory effect on the expression of PCSK9 mRNA in HELA cells. The highest inhibition rate was in the 35 / 75 group, reaching 68.9%, which was higher than the positive control; the inhibition rates of the 6 / 46, 32 / 72 and 40 / 80 groups also reached 63.0%, 64.8% and 62.5%, respectively, and the inhibition rates of the 14 / 54, 20 / 60, 29 / 69, 31 / 71, 33 / 73 and 38 / 78 groups were also above 50%.

[0142] 1. Experimental Materials

[0143] Unmodified oligonucleotides: unmodified oligonucleotide sequences 1-80 listed in Table 2, synthesized in Example 1

[0144] Cell type: HELA cells

[0145] Positive control: unmodified PC (Inclisiran drug sequence from Alnylam) sense strand and antisense strand (sequences 81 and 82 in Table 2), synthesized in Example 1

[0146] Drug solvent: sterile enzyme-free water, Gibco DMEM

[0147] 2. Experimental Methods

[0148] qRT-PCR was used to detect the inhibitory effect of the samples on the expression of PCSK9 gene mRNA in HELA cell lines.

[0149] 2.1 Cell culture

[0150] Take the HELA cell line in subculture, and place the logarithmically growing cells in 10% fetal bovine serum RPMI 1640 culture medium (supplemented with 100× penicillin and streptomycin 10 μL / mL) in a cell culture incubator at 37°C with 5% CO2, and change the medium once a day. Digest and subculture with 0.25% trypsin, centrifuge at 1000r / min for 5 minutes, discard the supernatant, and add fresh culture medium for subculture.

[0151] 2.2 Cell transfection

[0152] Preparation of transfection mixture: Mix Lipofectamine RNAiMAX and Opti-MEM in a ratio of 1.5:98.5 and vortex to mix.

[0153] Preparation of transfection reagent: Add 60 μL of siRNA solution diluted in Opti-MEM to 60 μL of transfection mixture at a ratio of 1:1 (v / v), vortex and let stand at room temperature for 15 minutes to obtain lipid nanoparticles (LNP). Take 12.5 μL for encapsulation efficiency detection.

[0154] Transfection reagent for blank control group: Add 60 μL of prepared transfection mixture to 60 μL of Opti-MEM. Vortex to mix well and let stand at room temperature for 15 minutes.

[0155] 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 cells). After mixing using the cross method, the cells were placed in a 37°C, 5% CO2 cell culture incubator and cultured for 40 h.

[0156] 2.3 PCSK9 mRNA detection

[0157] 1) RNA extraction

[0158] a. Aspirate the culture medium in the 12-well plate, add 0.5 mL 1× PBS to each well to wash the cells, and aspirate the PBS. Add 0.5 mL TRIzol reagent to the wells, blow the cells with the pipette tip to fully lyse, transfer to a 1.5 mL RNase-free EP tube, and let stand at room temperature for 5 minutes.

[0159] 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 take 200 μL of the supernatant into a new EP tube.

[0160] c. Add an equal volume of isopropanol, gently mix the liquid in the tube by inverting it upside down, let it stand at -20℃ for 10 minutes, centrifuge at 4℃, 12,000×g for 15 minutes, and discard the supernatant.

[0161] d. Add 0.5 mL of 75% ethanol, gently wash the RNA precipitate, centrifuge at 12,000 × g at 4°C for 5 min, and remove the supernatant. Repeat the rinse once, centrifuge at 12,000 × g at 4°C for 1 min, and remove the residual ethanol with a micropipette tip.

[0162] e. Dry the residual ethanol at room temperature for 2-3 minutes, add 40 μL RNase-free ddH2O and dissolve.

[0163] 2) RNA concentration detection

[0164] Use nanodrop to test RNA concentration. Use 2μL RNase-free ddH2O as blank control and 2μL RNA sample for each test. Record the sample concentration.

[0165] 3) PCSK9 mRNA quantitative detection

[0166] In a 15 mL centrifuge tube, add the remaining components except primers and template in 7.5×84=630 portions, marked as A.

[0167] 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).

[0168] The upstream and downstream primers of the internal reference gene GAPDH and the target gene PCSK9 are mixed and set aside (marked as C).

[0169] Add 1 μL of B1 and 1 μL of C to each well of the PCR plate, cover with a sealing film, centrifuge at 3000 rpm for 1 min, and put on the machine.

[0170] *This step is performed on ice to maintain low temperature conditions.

[0171] Place the plate into the qPCR instrument and run the following program:

[0172] Reverse transcription: 55°C, 15 min;

[0173] Pre-denaturation: 95°C, 30 sec;

[0174] Cyclic reaction: 95°C, 10 sec; 60°C, 35 sec; 40 cycles;

[0175] Melting curve: 95℃, 15sec; 60℃, 60sec; 95℃, 15sec.

[0176] The running time is about 2h. Analyze the experimental results and calculate 2-ΔΔCt.

[0177] 2.4 Data Processing

[0178] The PCSK9 mRNA expression rate (%) was calculated as follows:

[0179] Expression rate = (PCSK9 mRNA expression level / PCSK9 mRNA expression level in blank control group) × 100%;

[0180] The PCSK9 gene expression inhibition rate (%) was calculated as follows:

[0181] PCSK9 gene expression inhibition rate = 1-expression rate (%).

[0182] 3. Experimental results

[0183] Specific data on the inhibitory effect of synthetic unmodified oligonucleotides on PCSK9 gene expression are shown in Tables 4-7.

[0184] Different sequences have very different inhibition rates on PCSK9 gene expression, ranging from 0-65%. Generally speaking, sequences with inhibition rates higher than 48% can be optimized as lead compounds, while sequences with inhibition rates between 30-48% have little significance for optimization, and sequences with inhibition rates below 30% have very poor effects and will not be considered.

[0185] 1) Some unmodified sequences had significant inhibitory effects on PCSK9 gene expression, including double-stranded siRNA paired with 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80. Among them, the strongest inhibitory effects were found in the 32 / 72 and 35 / 75 groups, with inhibition rates of 64.8% and 68.9%, respectively.

[0186] Table 4 Inhibition rate of PCSK9 gene expression by unmodified sequence -1

[0187]

[0188]

[0189] As can be seen from Table 4, the double-stranded siRNA paired with unmodified sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 had a significant inhibitory effect on the expression of PCSK9 mRNA in HELA cells, among which the strongest inhibitory effects were in the 32 / 72 and 35 / 75 groups, with inhibition rates of 64.8% and 68.9%, respectively, which were higher than those in the positive control group; the inhibition rates of the 6 / 46 and 40 / 80 groups were also 63.0% and 62.5%, respectively; the inhibition rates of the 14 / 54, 20 / 60, 29 / 69, 31 / 71, 33 / 73 and 38 / 78 groups were also above 50%.

[0190] The above compounds have significant inhibitory effects on PCSK9 gene expression, with inhibition rates of more than 50%, and can be optimized as lead compounds.

[0191] 2) Some unmodified sequences had a strong inhibitory effect on PCSK9 gene expression, with inhibition rates ranging from 30-48%, including paired double-stranded siRNAs such as 4 / 44, 10 / 50 and 13 / 53.

[0192] Table 5 Inhibition rate of PCSK9 gene expression by unmodified sequence -2

[0193]

[0194] The inhibition rates of PCSK9 gene expression of each sequence in Table 5 were between 30-48%. The inhibition rates of different sequences varied greatly, for example, the inhibition rates of 4 / 44, 10 / 50, 34 / 74, 37 / 77 and 39 / 79 groups were all above 45%, while the inhibition rates of 23 / 63 and 36 / 76 groups were below 35%.

[0195] 3) Some unmodified sequences had poor inhibitory effects on PCSK9 gene expression, with inhibition rates ranging from 1-30%, including paired double-stranded siRNAs such as 2 / 42, 3 / 43 and 5 / 45.

[0196] Table 6 Inhibition rate of PCSK9 gene expression by unmodified sequence -3

[0197]

[0198] The inhibition rates of PCSK9 gene expression of each sequence in Table 6 were between 1-30%. The inhibition rates of different sequences varied greatly, for example, the inhibition rates of 21 / 61 and 24 / 64 groups were both above 25%, while the inhibition rates of 11 / 51 and 25 / 65 groups were below 5%.

[0199] 4) Some sequences had no inhibitory effect on PCSK9 gene expression, including paired double-stranded siRNAs such as 1 / 41, 7 / 47 and 8 / 48.

[0200] Table 7 Inhibition rate of PCSK9 gene expression by unmodified sequence -4

[0201]

[0202] As shown in Table 7, double-stranded siRNAs paired with 1 / 41, 7 / 47, 8 / 48, 9 / 49, 16 / 56, 17 / 57, 19 / 59, 26 / 66, 27 / 67 and 28 / 68 had no inhibitory effect on PCSK9 gene expression.

[0203] From this, we can see that not all siRNA sequences designed for PCSK9 mRNA sequences have inhibitory activity on PCSK9 mRNA expression. Different sequences can have very large differences. It takes a lot of creative work to screen out siRNA sequences that have significant inhibitory effects on PCSK9 mRNA.

[0204] The double-stranded siRNA paired with unmodified oligonucleotides 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 designed in the present invention can significantly inhibit the expression of PCSK9 gene, thereby inhibiting the concentration of cholesterol in the blood, thereby achieving the treatment of hypercholesterolemia.

[0205] Example 3: Inhibitory effect of alternatively modified oligonucleotides on the PCSK9 gene

[0206] The inventors adopted a 2′-F and 2′-OMe alternating modification scheme for the unmodified oligonucleotide sequence 1-80 designed in the present disclosure to investigate the effect of such modification on the activity of the unmodified oligonucleotide sequence 1-80.

[0207] The activity test results showed that the inhibition rate of PCSK9 gene by double-stranded siRNA paired with 2'-OMe and 2'-F alternating modification sequences 88 / 128, 114 / 154 and 122 / 162 reached more than 70%, which was equivalent to the inhibition rate of 163 / 164; the inhibition rates of 102 / 142, 115 / 155 and 117 / 157 groups were also relatively high, reaching more than 60%; the inhibition rates of 96 / 136, 111 / 151, 113 / 153 and 120 / 160 groups were also all above 50%.

[0208] At the same time, it was found that the alternating modification of 2'-F and 2'-OMe had different effects on the activity of different sequences. Some sequences could significantly increase their inhibition rate, some sequences had reduced inhibition rate after modification, and some sequences had basically no effect on inhibition rate. Therefore, it can be seen that the alternating modification scheme of 2'-OMe and 2'-F has inconsistent effects on different sequences, and not all sequences can improve inhibition after the same modification.

[0209] 1. Experimental Materials

[0210] Alternating modified oligonucleotides: The modified oligonucleotides listed in Table 3 were modified with 2'-OMe and 2'-F alternatingly, wherein the odd-numbered positions of the sense strand and the even-numbered positions of the antisense strand were all modified with 2'-F, and the even-numbered positions of the sense strand and the odd-numbered positions of the antisense strand were all modified with 2'-OMe, and were synthesized in Example 1.

[0211] Cell type: HELA cells

[0212] Drug solvent: sterile enzyme-free water, Gibco DMEM.

[0213] 2. Experimental Methods

[0214] The experimental method is the same as that in Example 2.

[0215] 3. Experimental results

[0216] Specific data on the inhibitory effect of alternatively modified oligonucleotides on PCSK9 gene expression are shown in Tables 8-10.

[0217] 1) Some alternatively modified sequences have a significant inhibitory effect on PCSK9 gene expression, including double-stranded siRNAs paired with 122 / 162, 114 / 154, 88 / 128, 102 / 142, 115 / 155, 117 / 157, 113 / 153, 120 / 160, 111 / 151 and 96 / 136. Among them, the inhibition rates of 88 / 128, 114 / 154 and 122 / 162 groups all reached 70%, and the inhibitory effects of these three groups of sequences were significantly improved compared with the unmodified sequences.

[0218] Table 8 Inhibition rate of PCSK9 gene expression by alternating modified sequences -1

[0219]

[0220]

[0221] As can be seen from Table 8, double-stranded siRNAs paired with 2'-OMe and 2'-F alternating modified sequences 88 / 128, 96 / 136, 102 / 142, 111 / 151, 113 / 153, 114 / 154, 115 / 155, 117 / 157, 120 / 160 and 122 / 162 can significantly inhibit the expression of PCSK9 gene, and the inhibition rate is more than 50%. Among them, the inhibition rates of 88 / 128, 114 / 154 and 122 / 162 groups reached 70%, which is equivalent to 163 / 164; the inhibition rates of 102 / 142, 115 / 155 and 117 / 157 groups are also relatively high, reaching 60%; the inhibition rates of 96 / 136, 111 / 151, 113 / 153 and 120 / 160 groups are also above 50%.

[0222] It can be seen from this that double-stranded siRNA paired with alternating modified sequences 88 / 128, 96 / 136, 102 / 142, 111 / 151, 113 / 153, 114 / 154, 115 / 155, 117 / 157, 120 / 160 and 122 / 162 can inhibit the expression of the PCSK9 gene, thereby inhibiting the concentration of cholesterol in the blood, and can be used to treat hypercholesterolemia.

[0223] Furthermore, the unmodified sequences corresponding to the above-mentioned alternating modified sequences, i.e., double-stranded siRNAs paired with 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80, also have a significant inhibitory effect on the expression of the PCSK9 gene, with the inhibition rate being above 50%. This indicates that the sequences designed by the present disclosure, whether unmodified or modified with 2'-OMe and 2'-F alternatingly, can inhibit the expression of the PCSK9 gene.

[0224] By comparing the inhibition rate with that of the unmodified sequence, it can be seen that the alternating modification has different effects on the activity of different sequences. Some sequences can significantly improve the inhibition rate, for example, the inhibition rates of the unmodified sequence 6 / 46 and 40 / 80 groups are 63.0% and 62.5%, respectively, and the inhibition rates of the modified 88 / 128 and 122 / 162 groups are 70.0% and 70.9%, respectively, which are 7.0% and 8.4% higher than those without modification; the inhibition rate of some sequences decreases after modification, for example, the inhibition rates of the unmodified 14 / 54 and 35 / 75 groups are 56.2% and 68.9%, respectively, and the inhibition rates of the modified 96 / 136 and 117 / 157 groups are 52.8% and 60.2%, respectively, which are 3.4% and 8.7% lower than those without modification; some sequences have no effect on activity after modification, for example, the inhibition rate of the unmodified sequence 38 / 78 group is basically unchanged compared with the modified 120 / 160 group.

[0225] 2) The inhibition rate of PCSK9 gene expression by some alternatively modified sequences was between 40-48%, including 4 / 44, 11 / 51 and 16 / 56 paired double-stranded siRNAs.

[0226] Table 9 Inhibition rate of PCSK9 gene expression by alternating modified sequences -2

[0227]

[0228] As shown in Table 9, the inhibition rates of PCSK9 gene expression in the alternating modified sequence groups of 4 / 44, 11 / 51, 16 / 56, 17 / 57, 18 / 58, 19 / 59, 21 / 61, 22 / 62, 24 / 64, 26 / 66 and 37 / 77 were between 40% and 48%.

[0229] By comparing with the unmodified sequence, it can be seen that the alternating modification of 2'-OMe and 2'-F has different effects on the inhibition rate of different sequences. The inhibitory activity of some sequences is significantly improved. For example, the unmodified sequences 16 / 56, 17 / 57, 26 / 66 and 19 / 59 have no inhibitory effect on PCSK9 gene expression. After alternating modification, the inhibition rates of 98 / 138, 99 / 139, 108 / 148 and 101 / 141 can reach 45.5%, 40.6%, 40.8% and 40.3%, respectively. The activity of some sequences is reduced after modification. For example, the inhibition rate of the unmodified sequence 4 / 44 group is 46.4%. After alternating modification, the inhibition rate of the 86 / 126 group is 40.4%, which is 6.0% lower than that of the unmodified sequence. The alternating modification of some sequences has no effect on the activity. For example, the inhibition rate of the unmodified 37 / 77 group is not much different from that of the modified 119 / 159 group.

[0230] 3) The inhibition rate of PCSK9 gene expression by some alternatively modified sequences was between 30-40%, including 84 / 124, 87 / 127 and 90 / 130 paired double-stranded siRNAs.

[0231] Table 10 Inhibition rate of PCSK9 gene expression by alternating modified sequences-3

[0232]

[0233]

[0234] The alternative modified sequences listed in Table 10 have a certain inhibitory effect on PCSK9 gene expression, with an inhibition rate between 30-40%. The highest inhibition rates are in groups 112 / 152 and 116 / 156, which are 38.9% and 39.6%, respectively.

[0235] By comparing with the unmodified sequence, it can be seen that the alternating modification of 2'-OMe and 2'-F has different effects on the activity of different sequences. The activity of some sequences is improved. For example, the double-stranded siRNA paired with the unmodified sequences 8 / 48, 9 / 49 and 27 / 67 has no inhibitory effect on PCSK9mRNA expression. After alternating modification, the inhibition rates of 90 / 130, 91 / 131 and 109 / 149 groups reached 32.8%, 35.5% and 36.8%, respectively. The activity of some sequences is reduced. For example, the inhibition rates of the unmodified sequences 10 / 50 and 39 / 79 groups were 46.7% and 48.9%, respectively. After alternating modification, the inhibition rates of 92 / 132 and 121 / 161 groups were 35.4% and 36.3%, respectively, which were 11.3% and 12.6% lower than those without modification. The activity of some sequences had no effect after alternating modification. For example, the inhibition rate of the unmodified sequences 23 / 63 and 30 / 70 groups was basically unchanged compared with the modified sequences 105 / 145 and 112 / 152 groups.

[0236] 4) Some alternatively modified sequences have poor inhibitory effects on PCSK9 gene expression, such as 83 / 123, 85 / 125 and 89 / 129 paired double-stranded siRNA, with inhibition rates below 30%.

[0237] Table 11 Inhibition rate of PCSK9 gene expression by alternating modified sequences-4

[0238]

[0239] The 2'-OMe and 2'-F alternating modification sequences listed in Table 11 had poor inhibitory effects on PCSK9 gene expression, with inhibition rates all below 30%, and only 16.8% in the 110 / 150 group.

[0240] By comparing with the unmodified sequence, it can be seen that the alternating modification of 2'-OMe and 2'-F has different effects on the activity of different sequences. The activity of some sequences is improved. For example, the unmodified sequences 1 / 41, 7 / 47 and 28 / 68 have no inhibitory effect on PCSK9 mRNA expression. After alternating modification, the inhibition rates of 83 / 123, 89 / 129 and 110 / 150 are 25.4%, 23.5% and 16.8%, respectively; the activity of some sequences is reduced. For example, the inhibition rates of the unmodified sequences 13 / 53 and 36 / 76 are 41.8% and 33.8%, respectively. After alternating modification, the inhibition rates of 95 / 135 and 118 / 158 are 24.6% and 28.6%, respectively, which are 17.2% and 5.2% lower than those without modification.

[0241] It can be seen that the oligonucleotide sequence designed for PCSK9 mRNA disclosed in the present invention has a significant inhibitory effect on PCSK9 gene expression after being modified alternately with 2'-OMe and 2'-F, including double-stranded siRNAs paired with 88 / 128, 96 / 136, 102 / 142, 111 / 151, 113 / 153, 114 / 154, 115 / 155, 117 / 157, 120 / 160 and 122 / 162. Among them, the highest inhibition rate is 88 / 128, 114 / 154 and 122 / 162 groups, the inhibition rate of which reaches 70%, which is equivalent to 163 / 164, and the inhibition rate of other sequences is also above 50%. It can be used to develop drugs for treating diseases related to PCSK9 gene.

[0242] Moreover, the alternating modification of 2'-OMe and 2'-F has inconsistent effects on different sequences of the unmodified oligonucleotide sequences 1-80 designed in the present invention. The activities of some sequences are improved, some sequences are reduced, and some sequences have no change in activity. Not all sequences can improve their inhibitory effects after alternating modification, and there will be very large differences between different sequences.

[0243] Example 4: Comparison of inhibition rates between the designed sequences disclosed herein and similar sequences disclosed in the prior art

[0244] The 10 sequences with the highest inhibitory effects on PCSK9 gene expression in the present disclosure were compared with similar sequences disclosed in the prior art. The results showed that the unmodified sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 in the present disclosure were superior to similar sequences in the disclosed patents in terms of inhibitory effects on PCSK9 mRNA expression in HELA cells at the same concentration. For example, the inhibition rate of the sequence 32 / 72 disclosed in the present disclosure was increased by 20.3% over the similar sequence 170 / 180 disclosed in the prior art, the sequence 6 / 46 was increased by 22.6% over the similar sequence 165 / 175, and the sequence 40 / 80 was increased by 23.9% over the similar sequence 174 / 184.

[0245] 1. Experimental Materials

[0246] Sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 in the present disclosure, as well as sequences disclosed in the prior art that are close to these sequences, include sequences 165 / 175, 166 / 176, 167 / 177, 168 / 178, 169 / 179, 170 / 180, 171 / 181, 172 / 182, 173 / 183 and 174 / 184, as shown in Table 12.

[0247]

[0248] Some of the sequences disclosed in the patents listed in Table 12 have no modification at the 3' end, and some have TT modification or TsT modification at the 3' end. In order to compare the difference in inhibitory activity of the sequences designed in the present disclosure with the sequences disclosed in the prior art on the PCSK9 gene, the 3' ends of all sequences were modified in the same way. That is, the sequences disclosed in the patents with no modification at the 3' end were modified with TsT, and the sequences with TT modification at the 3' end were removed from the TT modification, and then modified with TsT, to obtain sequences 165-174 (sense strand) and 175-184 (antisense strand). These sequences were synthesized, and the specific synthesis method was the same as in Example 1.

[0249] 2. Experimental Methods

[0250] The inhibition rate of the oligonucleotides listed in Table 12 on the PCSK9 gene was detected, and the specific experimental method was the same as Example 2.

[0251] 3. Experimental results

[0252] The inhibitory activity of the sequences in the present disclosure and the sequences similar to those disclosed in the patents on PCSK9 gene expression is shown in Table 13.

[0253] Table 13 Comparison of the activity of the designed sequences disclosed in the present invention and similar sequences disclosed in the prior art

[0254]

[0255] As can be seen from Table 13, the unmodified sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 screened in the present disclosure have better inhibitory effects on PCSK9 gene expression than similar sequences in disclosed patents at the same concentration. For example, the inhibition rate of the sequence 32 / 72 disclosed in the present invention is 64.8%, while the inhibition rate of the similar sequence 170 / 180 disclosed in CN101484588B is only 46.9%, and the inhibition rate of sequence 32 / 72 is 17.9% higher than that of 170 / 180; the inhibition rate of the sequence 6 / 46 disclosed in the present invention is 63.0%, which is 18.9% higher than that of the similar sequence 165 / 175 disclosed in CN102124107A; the inhibition rate of the sequence 40 / 80 disclosed in the present invention is 62.5%, which is 20.1% higher than that of the similar sequence 174 / 184 disclosed in CN102124107A.

[0256] It can be seen from this that the inhibition rates of PCSK9 gene expression by oligonucleotides with similar sequences are not necessarily close, but they are likely to be very different. For example, compared with sequence 165 in CN102124107A, sequence 6 disclosed in the present invention only has 2 more nucleotides (UU) at the 3' end, but the inhibition rate is increased by 18.9%; compared with the sequence in CN102124107A (sequence 174), sequence 40 disclosed in the present invention only has 3 more nucleotides (CUU) at the 5' end and 3 more nucleotides (ATT) at the 3' end, but the inhibition rate is increased by 20.1%; compared with the sequence in CN101484588B (sequence 169), sequence 31 disclosed in the present invention only has 6 more nucleotides (AGUCAG) at the 3' end, but the inhibition rate is increased by 21.4%. Although these sequences in the present disclosure are very different from the sequences disclosed in the prior art, the inhibition rate of PCSK9 gene expression is significantly improved.

[0257] The present invention designs 80 small interfering RNA (siRNA) sequences for PCSK9 mRNA sequence, including 40 sense strands and 40 antisense strands, and performs 2'-OMe and 2'-F alternating modifications on these sequences. By testing the inhibitory activity of unmodified sequences and alternating modified sequences on PCSK9 gene expression, some target sequences with significant inhibitory effect on PCSK9 gene expression were screened out.

[0258] 1. Among the 80 unmodified oligonucleotide sequences designed, the inhibitory activities of different sequences on PCSK9 mRNA expression were very different. Some of them had significant inhibitory effects on PCSK9 gene expression, including paired double-stranded siRNAs such as 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80. Among them, the inhibition rates of 32 / and 72, 35 / 75 groups reached 64.8% and 68.9%, respectively, which were higher than the positive control; the inhibition rates of 6 / 46 and 40 / 80 groups also reached 63.0% and 62.5%, respectively, and the inhibition rates of 14 / 54, 20 / 60, 29 / 69, 31 / 71, 33 / 73 and 38 / 78 groups were also above 50%. Some sequences had high activity, such as 39 / 79, 10 / 50 and 4 / 44, which were between 30-48%. Some sequences had low activity, such as 2 / 42, 3 / 43 and 5 / 45, which were between 1-30%. Some sequences had no inhibitory effect, including 7 / 47, 9 / 49 and 19 / 59.

[0259] From this, we can see that not all siRNA sequences designed for PCSK9 mRNA sequences have inhibitory activity on PCSK9 gene expression. Different sequences can have very large differences. It takes a lot of creative work to screen out siRNA sequences that have a significant inhibitory effect on PCSK9 gene expression.

[0260] 2. The 80 designed siRNA sequences were modified with 2'-F and 2'-Me alternately. The activity test results showed that the inhibitory activity of different sequences with alternating modifications on PCSK9 gene expression was very different. Some of the alternating modified sequences had a significant inhibitory effect on PCSK9 gene expression, including 96 / 136, 102 / 142, 111 / 151, 113 / 153, 114 / 154, 115 / 155, 117 / 157, 120 / 160 and 122 / 162 paired double-stranded siRNA. Among them, 88 / 128, 114 / 154 and 122 / 162 had an inhibition rate of 70% on the target gene, which was equivalent to 163 / 164. The inhibition rates of other groups were also high, all above 50%. Some alternating modified sequences had high activity, such as 86 / 126, 93 / 133 and 98 / 138, with inhibition rates between 30-48%. Some alternatively modified sequences had lower activities, such as 83 / 123, 85 / 125 and 89 / 129, with inhibition rates below 30%.

[0261] It can be seen from this that not all 2'-F and 2'-Me alternating modification sequences have strong inhibitory activity on PCSK9 gene expression. Different sequences can have very large differences. It takes a lot of creative work to screen out the alternating modification sequences that have a significant inhibitory effect on PCSK9 gene expression.

[0262] 3. The 80 siRNA sequences designed had different effects on the activity of different sequences after 2'-F and 2'-Me were modified alternately. Some of them could significantly improve their inhibitory effects. For example, the unmodified sequences 16 / 56, 17 / 57 and 19 / 59 had no inhibitory effect on the expression of PCSK9 mRNA. After alternating modification, the inhibition rates of 98 / 138, 99 / 139 and 101 / 141 were 45.5%, 40.6% and 40.3%, respectively. Some of them had significantly reduced inhibition rates after alternating modification. For example, the unmodified sequences 10 / 50, 13 / 53 and 39 / 79 had inhibition rates of 46.7%, 41.8% and 48.9%, respectively. After alternating modification, the inhibition rates of 92 / 132, 95 / 135 and 121 / 161 were reduced by 11.3%, 17.2% and 12.6%, respectively. In addition, there are some sequences whose activities remain basically unchanged after modification. For example, the inhibition rate of the unmodified sequences 23 / 63 and 30 / 70 groups remains basically unchanged compared with the modified sequences 105 / 145 and 112 / 152 groups.

[0263] 4. The unmodified sequences 6 / 46, 14 / 54, 20 / 60, 29 / 69, 31 / 71, 32 / 72, 33 / 73, 35 / 75, 38 / 78 and 40 / 80 screened in the present disclosure have better inhibitory effects on PCSK9 gene expression than similar sequences in disclosed patents at the same concentration. For example, the inhibition rate of the sequence 32 / 72 disclosed in the present invention is 64.8%, while the inhibition rate of the similar sequence 170 / 180 disclosed in CN101484588B is only 46.9%, and the inhibition rate of sequence 32 / 72 is 17.9% higher than that of 170 / 180; the inhibition rate of the sequence 6 / 46 disclosed in the present invention is 63.0%, which is 18.9% higher than that of the similar sequence 165 / 175 disclosed in CN102124107A; the inhibition rate of the sequence 40 / 80 disclosed in the present invention is 62.5%, which is 20.1% higher than that of the similar sequence 174 / 184 disclosed in CN102124107A.

[0264] The results show that the inhibition rates of PCSK9 gene expression by oligonucleotides with similar sequences are not necessarily close, but may be very different. Although the sequences disclosed in the present disclosure are slightly different from the sequences disclosed in the prior art, the inhibition rates of PCSK9 gene expression are significantly improved.

[0265] The present disclosure screened out some siRNA sequences with significant inhibitory activity on PCSK9 gene expression through unique design and a lot of creative work. The series of siRNA sequences designed by the present disclosure, including unmodified and modified sequences, can be used to develop drugs for treating related diseases caused by high cholesterol.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for reducing the expression of PCSK9, wherein the oligonucleotide is an oligonucleotide duplex consisting of a modified sequence of the sense strand as shown in SEQ ID NO. 40 and a modified sequence of the antisense strand as shown in SEQ ID NO. 80: the modified sequence of the sense strand is the sequence shown in SEQ ID NO. 122; the modified sequence of the antisense strand is the sequence shown in SEQ ID NO. 162, wherein: The sequence shown in SEQ ID NO. 40 is as follows: CUUUUGUAACUUGAAGAUAUUUATTTsT; The sequence shown in SEQ ID NO. 80 is as follows: AAUAAAUAUCUUCAAGUUACAAAAGCATsT; The sequence shown in SEQ ID NO. 122 is as follows: CfUmUfUmUfGmUfAmAfCmUfUmGfAmAfGmAfUmAfUmUfUmAfTmTfTsT; The sequence shown in SEQ ID NO. 162 is as follows: AmAfUmAfAmAfUmAfUmCfUmUfCmAfAmGfUmUfAmCfAmAfAmAfGmCfAmTsT.

2. A conjugate for reducing the expression of PCSK9, comprising the oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, and a targeting ligand conjugated thereto.

3. The conjugate of claim 2, wherein the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid.

4. The conjugate of claim 2 or 3, wherein the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

5. The conjugate of claim 4, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

6. A composition comprising the oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof or the conjugate according to any one of claims 2 to 5, and optionally a pharmaceutically acceptable carrier.

7. Use of the oligonucleotide or pharmaceutically acceptable salt thereof according to claim 1, the conjugate according to any one of claims 2 to 5, or the composition according to claim 6 in the preparation of a medicament for treating or / and preventing a disease associated with the expression of the PCSK9 gene, wherein the disease is a lipid disorder.

8. The use according to claim 7, wherein the disease in the lipid disorder is hyperlipidemia, atherosclerosis and / or one or more symptoms or one or more symptoms of complications thereof.

9. The use according to claim 8, wherein the hyperlipidemia is hypercholesterolemia.

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